Selective BACE1 inhibitors

ABSTRACT

The present invention provides a compound of Formula II: 
                         
or a pharmaceutically acceptable salt thereof, that are inhibitors of BACE.

The present invention relates to certain novel selective BACE1inhibitors, to pharmaceutical compositions comprising the compounds, tomethods of using the compounds to treat physiological disorders, and tointermediates and processes useful in the synthesis of the compounds.

The present invention is in the field of treatment of Alzheimer'sdisease and other diseases and disorders involving amyloid β (Abeta)peptide, a neurotoxic and highly aggregatory peptide segment of theamyloid precursor protein (APP). Alzheimer's disease is a devastatingneurodegenerative disorder that affects millions of patients worldwide.In view of the currently approved agents on the market which afford onlytransient, symptomatic benefits to the patient rather than halting,slowing, or reversing the disease, there is a significant unmet need inthe treatment of Alzheimer's disease.

Alzheimer's disease is characterized by the generation, aggregation, anddeposition of Abeta in the brain. Complete or partial inhibition ofβ-secretase (β-site amyloid precursor protein-cleaving enzyme; BACE) hasbeen shown to have a significant effect on plaque-related andplaque-dependent pathologies in mouse models suggesting that even smallreductions in Abeta peptide levels might result in a long-termsignificant reduction in plaque burden and synaptic deficits, thusproviding significant therapeutic benefits, particularly in thetreatment of Alzheimer's disease. In addition, two homologs of BACE havebeen identified which are referred to as BACE1 and BACE2, and it isbelieved that BACE1 is the most clinically important to development ofAlzheimer's disease. BACE1 is mainly expressed in the neurons whileBACE2 has been shown to be expressed primarily in the periphery. (See D.Oehlrich, Bioorg. Med. Chem. Lett., 24, 2033-2045 (2014)) In addition,BACE2 may be important to pigmentation as it has been identified asplaying a role in the processing of pigment cell-specific melanocyteprotein (See L. Rochin, et al., Proc. Natl. Acad. Sci. USA, 110(26),10658-10663 (2013)). BACE inhibitors with central nervous system (CNS)penetration, particularly inhibitors that are selective for BACE1 overBACE2 are desired to provide treatments for Abeta peptide-mediateddisorders, such as Alzheimer's disease.

U.S. Pat. No. 8,158,620 discloses fused aminodihydrothiazine derivativeswhich possess BACE1 inhibitory activity and are further disclosed asuseful therapeutic agents for a neurodegenerative disease caused by Aβpeptide, such as Alzheimer's type dementia. In addition, U.S. Pat. No.8,338,407 discloses certain fused aminodihydrothiazine derivativeshaving BACE1 inhibitory effect useful in treating certainneurodegenerative diseases, such as Alzheimer-type dementia.

The present invention provides certain novel compounds that areinhibitors of BACE. In addition, the present invention provides certainnovel compounds that are selective inhibitors of BACE1 over BACE2.Furthermore, the present invention provides certain novel compoundswhich penetrate the CNS. The present invention also provides certainnovel compounds which have the potential for an improved side-effectprofile, for example through selective inhibition of BACE1 over BACE2.

Accordingly, the present invention provides a compound of Formula I:

or a pharmaceutically acceptable salt thereof.

In addition, the present invention provides a compound of Formula Ia:

or a pharmaceutically acceptable salt thereof.

The present invention further provides a compound of Formula II:

wherein R is methyl, ethyl, or cyclopropyl;or a pharmaceutically acceptable salt thereof.

In addition, the present invention provides a compound of Formula IIa:

or a pharmaceutically acceptable salt thereof.

The present invention also provides a method of treating Alzheimer'sdisease in a patient, comprising administering to a patient in need ofsuch treatment an effective amount of a compound of Formulas I, Ia, II,or IIa, or a pharmaceutically acceptable salt thereof.

The present invention further provides a method of treating theprogression of mild cognitive impairment to Alzheimer's disease in apatient, comprising administering to a patient in need of such treatmentan effective amount of a compound of Formulas I, Ia, II, or IIa, or apharmaceutically acceptable salt thereof. The present invention alsoprovides a method of inhibiting BACE in a patient, comprisingadministering to a patient in need of such treatment an effective amountof a compound of Formulas I, Ia, II, IIa, or a pharmaceuticallyacceptable salt thereof. The present invention also provides a methodfor inhibiting BACE-mediated cleavage of amyloid precursor protein,comprising administering to a patient in need of such treatment aneffective amount of a compound of Formulas I, Ia, II, or IIa, or apharmaceutically acceptable salt thereof. The invention further providesa method for inhibiting production of Abeta peptide, comprisingadministering to a patient in need of such treatment an effective amountof a compound of Formulas I, Ia, II, or IIa, or a pharmaceuticallyacceptable salt thereof.

Furthermore, this invention provides a compound of Formulas I, Ia, II,or IIa, or a pharmaceutically acceptable salt thereof for use intherapy, in particular for the treatment of Alzheimer's disease or forthe treatment of the progression of mild cognitive impairment toAlzheimer's disease. Even furthermore, this invention provides the useof a compound of Formulas I, Ia, II, or IIa, or a pharmaceuticallyacceptable salt thereof, for the manufacture of a medicament for thetreatment of Alzheimer's disease.

The invention further provides a pharmaceutical composition, comprisinga compound of Formulas I, Ia, II, or IIa, or a pharmaceuticallyacceptable salt thereof, with one or more pharmaceutically acceptablecarriers, diluents, or excipients. The invention further provides aprocess for preparing a pharmaceutical composition, comprising admixinga compound of Formulas I, Ia, II, or IIa, or a pharmaceuticallyacceptable salt thereof, with one or more pharmaceutically acceptablecarriers, diluents, or excipients. This invention also encompasses novelintermediates and processes for the synthesis of the compounds ofFormulas I, Ia, II, and IIa.

Mild cognitive impairment has been defined as a potential prodromalphase of dementia associated with Alzheimer's disease based on clinicalpresentation and on progression of patients exhibiting mild cognitiveimpairment to Alzheimer's dementia over time. (Morris, et al., Arch.Neurol., 58, 397-405 (2001); Petersen, et al., Arch. Neurol., 56,303-308 (1999)). The term “treating the progression of mild cognitiveimpairment to Alzheimer's disease” includes restraining, slowing,stopping, or reversing the progression of mild cognitive impairment toAlzheimer's disease in a patient.

As used herein, the terms “treating” or “to treat” includes restraining,slowing, stopping, or reversing the progression or severity of anexisting symptom or disorder.

As used herein, the term “patient” refers to a human.

The term “inhibition of production of Abeta peptide” is taken to meandecreasing of in vivo levels of Abeta peptide in a patient.

As used herein, the term “effective amount” refers to the amount or doseof compound of the invention, or a pharmaceutically acceptable saltthereof which, upon single or multiple dose administration to thepatient, provides the desired effect in the patient under diagnosis ortreatment.

An effective amount can be readily determined by the attendingdiagnostician, as one skilled in the art, by the use of known techniquesand by observing results obtained under analogous circumstances. Indetermining the effective amount for a patient, a number of factors areconsidered by the attending diagnostician, including, but not limitedto: the species of patient; its size, age, and general health; thespecific disease or disorder involved; the degree of or involvement orthe severity of the disease or disorder; the response of the individualpatient; the particular compound administered; the mode ofadministration; the bioavailability characteristics of the preparationadministered; the dose regimen selected; the use of concomitantmedication; and other relevant circumstances.

The compounds of the present invention are generally effective over awide dosage range. For example, dosages per day normally fall within therange of about 0.01 to about 20 mg/kg of body weight. In some instancesdosage levels below the lower limit of the aforesaid range may be morethan adequate, while in other cases still larger doses may be employedwith acceptable side effects, and therefore the above dosage range isnot intended to limit the scope of the invention in any way.

The compounds of the present invention are preferably formulated aspharmaceutical compositions administered by any route which makes thecompound bioavailable, including oral and transdermal routes. Mostpreferably, such compositions are for oral administration. Suchpharmaceutical compositions and processes for preparing same are wellknown in the art. (See, e.g., Remington: The Science and Practice ofPharmacy (D. B. Troy, Editor, 21st Edition, Lippincott, Williams &Wilkins, 2006).

The compounds of Formulas I, Ia, II, or IIa, or pharmaceuticallyacceptable salts thereof are particularly useful in the treatmentmethods of the invention, but certain groups, substituents, andconfigurations are preferred. The following paragraphs describe suchpreferred groups, substituents, and configurations. It will beunderstood that these preferences are applicable both to the treatmentmethods and to the new compounds of the invention.

Thus, the compound of Formulas I and II wherein the fused bicyclic ringis in the CIS configuration, or pharmaceutically acceptable saltthereof, is preferred. For example, one of ordinary skill in the artwill appreciate that the compound of Formula Ia is in the CIS relativeconfiguration for the centers labeled 1 and 2 as shown in Scheme Abelow. In addition, the compound of Formula Ia is comprised of a corethat contains three chiral centers at the carbon atoms labeled 1, 2, and3. It is understood by one of skill in the art, that the numberingsystem used in Scheme A may not correspond to the numbering system usedfor naming compounds herein, and as such, the numbering system used inScheme A is for illustrative purposes. The preferred relativeconfiguration for the three chiral centers of Formula Ia is shown inScheme A:

Further compounds of the present invention are:

-   N-[3-[(4aS,7aS)-2-amino-5-(1,1-difluoroethyl)-4,4a,5,7-tetrahydrofuro[3,4-d][1,3]thiazin-7a-yl]-4-fluoro-phenyl]-5-(1,2,4-triazol-1-yl)pyrazine-2-carboxamide;-   N-[3-[(4aSR,5SR,7aSR)-2-amino-5-(1,1-difluoroethyl)-4,4a,5,7-tetrahydrofuro[3,4-d][1,3]thiazin-7a-yl]-4-fluoro-phenyl]-5-(1,2,4-triazol-1-yl)pyrazine-2-carboxamide,    and pharmaceutically acceptable salts thereof.

Although the present invention contemplates all individual enantiomersand diasteromers, as well as mixtures of the enantiomers of saidcompounds, including racemates, the compounds with the absoluteconfiguration as set forth below are especially preferred:

-   N-[3-[(4aS,5S,7aS)-2-amino-5-(1,1-difluoroethyl)-4,4a,5,7-tetrahydrofuro[3,4-d][1,3]thiazin-7a-yl]-4-fluoro-phenyl]-5-(1,2,4-triazol-1-yl)pyrazine-2-carboxamide,    and the pharmaceutically acceptable salts thereof; and-   N-[3-[(4aS,5S,7aS)-2-amino-5-(1,1-difluoroethyl)-4,4a,5,7-tetrahydrofuro[3,4-d][1,3]thiazin-7a-yl]-4-fluoro-phenyl]-5-(1,2,4-triazol-1-yl)pyrazine-2-carboxamide    hydrate.

In addition,N-[3-[(4aS,5S,7aS)-2-amino-5-(1,1-difluoroethyl)-4,4a,5,7-tetrahydrofuro[3,4-d][1,3]thiazin-7a-yl]-4-fluoro-phenyl]-5-(1,2,4-triazol-1-yl)pyrazine-2-carboxamideis particularly preferred.

Furthermore,N-[3-[(4aS,5S,7aS)-2-amino-5-(1,1-difluoroethyl)-4,4a,5,7-tetrahydrofuro[3,4-d][1,3]thiazin-7a-yl]-4-fluoro-phenyl]-5-(1,2,4-triazol-1-yl)pyrazine-2-carboxamidemalonate; and

-   N-[3-[(4aS,5S,7aS)-2-amino-5-(1,1-difluoroethyl)-4,4a,5,7-tetrahydrofuro[3,4-d][1,3]thiazin-7a-yl]-4-fluoro-phenyl]-5-(1,2,4-triazol-1-yl)pyrazine-2-carboxamide    4-methylbenzenesulfonate are especially preferred.

One of ordinary skill in the art will appreciate that compounds of theinvention can exist in tautomeric forms, as depicted below in Scheme B.When any reference in this application to one of the specific tautomersof the compounds of the invention is given, it is understood toencompass both tautomeric forms and all mixtures thereof.

Additionally, certain intermediates described in the followingpreparations may contain one or more nitrogen or oxygen protectinggroups. It is understood that protecting groups may be varied asappreciated by one of skill in the art depending on the particularreaction conditions and the particular transformations to be performed.The protection and deprotection conditions are well known to the skilledartisan and are described in the literature (See for example “Greene'sProtective Groups in Organic Synthesis”, Fourth Edition, by Peter G. M.Wuts and Theodora W. Greene, John Wiley and Sons, Inc. 2007).

Individual isomers, enantiomers, and diastereomers may be separated orresolved by one of ordinary skill in the art at any convenient point inthe synthesis of compounds of the invention, by methods such asselective crystallization techniques or chiral chromatography (See forexample, J. Jacques, et al., “Enantiomers, Racemates, and Resolutions”,John Wiley and Sons, Inc., 1981, and E. L. Eliel and S. H. Wilen,“Stereochemistry of Organic Compounds”, Wiley-Interscience, 1994).

A pharmaceutically acceptable salt of the compounds of the invention,such as a hydrochloride salt, can be formed, for example, by reaction ofan appropriate free base of a compound of the invention, an appropriatepharmaceutically acceptable acid such as hydrochloric acid in a suitablesolvent such as diethyl ether under standard conditions well known inthe art. Additionally, the formation of such salts can occursimultaneously upon deprotection of a nitrogen protecting group. Theformation of such salts is well known and appreciated in the art. See,for example, Gould, P. L., “Salt selection for basic drugs,”International Journal of Pharmaceutics, 33: 201-217 (1986); Bastin, R.J., et al. “Salt Selection and Optimization Procedures forPharmaceutical New Chemical Entities,” Organic Process Research andDevelopment, 4: 427-435 (2000); and Berge, S. M., et al.,“Pharmaceutical Salts,” Journal of Pharmaceutical Sciences, 66: 1-19,(1977).

Certain abbreviations are defined as follows: “APP” refers to amyloidprecursor protein; “BSA” refers to Bovine Serum Albumin; “CDI” refers to1,1′-carbonyldiimidazole; “cDNA” refers to complementarydeoxyribonucleic acid; “DCC” refers to 1,3-dicyclohexylcarbodiimide;“Deoxo-Fluor®” refers to bis(2-methoxyethyl)aminosulfur trifluoride;“DIC” refers to 1,3-diisopropylcarbodiimide; “DMAP” refers to4-dimethylaminopyridine; “DMEM” refers to Dulbecco's Modified Eagle'sMedium; “DMSO” refers to dimethyl sulfoxide; “EBSS” refers to Earle'sBalances Salt Solution; “EDCI” refers to1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; “ELISA”refers to enzyme-linked immunosorbent assay; “F12” refers to Ham's F12medium; “FBS” refers to Fetal Bovine Serum; “Fc” refers to fragmentcrystallizable; “FLUOLEAD™” refers to4-tert-butyl-2,6-dimethylphenylsulfur trifluoride; “FRET” refers tofluorescence resonance energy transfer; “HATU” refers to(dimethylamino)-N,N-dimethyl(3H-[1,2,3]triazolo[4,5-b]pyridin-3-yloxy)methaniminiumhexafluorophosphate; “HBTU” refers to(1H-benzotriazol-1-yloxy)(dimethylamino)-N,N-dimethylmethaniminiumhexafluorophosphate; “HEK” refers to human embryonic kidney;“HF-pyridine” refers to hydrogen fluoride pyridine or Olah's reagent orpoly(pyridine fluoride); “HOAt” refers to 1-hydroxy-7-azabenzotriazole;“HOBT” refers to 1-hydroxylbenzotriazole hydrate; “IC₅₀” refers to theconcentration of an agent that produces 50% of the maximal inhibitoryresponse possible for that agent; “IgG₁” refers to immunoglobulin-likedomain Fc-gamma receptor; “MEM” refers to Minimum Essential Medium;“PBS” refers to phosphate buffered saline; “PDAPP” refers to plateletderived amyloid precursor protein; “Ph” refers to a phenyl group;“PyBOP” refers to (benzotriazol-1-yl-oxytripyrrolidinophosphoniumhexafluorophosphate); “PyBrOP” refers to bromo-tris-pyrrolidinophosphoniumhexafluorophosphate; “RFU” refers to relative fluorescenceunit; “RT-PCR” refers to reverse transcription polymerase chainreaction; “SDS-PAGE” refers to sodium dodecyl sulfate polyacrylamide gelelectrophoresis; “SCX” refers to strong cation exchange; “SFC” refers tosuper critical chromatography; “T3P®” refers to propylphosphonicanhydride; “THF” refers to tetrahydrofuran; “TEMPO” refers to(2,2,6,6-tetramethyl-piperidin-1-yl)oxyl; “TMEM” refers to transmembraneprotein; “trityl” refers to a group of the formula “(Ph)₃C-;“XtalFluor-E® or DAST difluorosulfinium salt” refers to(diethylamino)difluorosulfonium tetrafluoroborate orN,N-diethyl-S,S-difluorosulfiliminium tetrafluoroborate; and“XtalFluor-M® or morpho-DAST difluorosulfinium salt” refers todifluoro(morpholino)sulfonium tetrafluoroborate ordifluoro-4-morpholinylsulfonium tetrafluoroborate.

It is understood by one of ordinary skill in the art that the terms“tosylate”, “toluenesulfonic acid”, “p-toluenesulfonic acid”, and“4-methylbenzene sulfonic acid” refer to the compound of the followingstructure:

The compounds of the present invention, or salts thereof, may beprepared by a variety of procedures known to one of ordinary skill inthe art, some of which are illustrated in the schemes, preparations, andexamples below. One of ordinary skill in the art recognizes that thespecific synthetic steps for each of the routes described may becombined in different ways, or in conjunction with steps from differentschemes, to prepare compounds of the invention, or salts thereof. Theproducts of each step in the schemes below can be recovered byconventional methods well known in the art, including extraction,evaporation, precipitation, chromatography, filtration, trituration, andcrystallization. In the schemes below, all substituents unless otherwiseindicated, are as previously defined. The reagents and startingmaterials are readily available to one of ordinary skill in the art. Thefollowing schemes, preparations, and examples further illustrate theinvention.

In Scheme 1, step A, trimethylsulfonium iodide is treated with anorganic base such as n-butyllithium at a temperature of about −50° C. ina solvent such as THF. A protected oxymethyl oxirane, protected with asuitable protecting group, such as a trityl group, is then added to thebasic solution at −10° C. and allowed to stir for about 2 hours to givethe protected product of Scheme 1, Step A. “PG” is a protecting groupdeveloped for the amino group or oxygen group such as carbamates,amides, or ethers. Such protecting groups are well known and appreciatedin the art. The protected product of Step A is reacted with anα-haloester such as tert-butoxy bromoacetate using tetra-N-butylammoniumsulfate or other quaternary ammonium salt phase transfer catalysts in asolvent such as toluene and an aqueous inorganic base such as sodiumhydroxide at about room temperature to give the compound of Scheme 1,Step B. Such alkylations are well known in the art. Alternatively a basesuch as 60% sodium hydride in oil with solvents such asN,N-dimethylformamide or THF and a temperature range of 0 to 100° C. canbe used to give the protected product of Step B. The tert-butoxycarbonyl acetate is converted to an oxime over a 2-step procedure. Areducing agent such as isobutylaluminum hydride in hexanes is addeddropwise at a temperature of about −70° C. followed by the dropwiseaddition of an aqueous acid such as hydrochloric acid at a temperatureof about −60° C. The work-up is accomplished with an organic extractionto give the intermediate material. This material is dissolved in anorganic solvent such as dichloromethane and treated with sodium acetatefollowed by hydroxylamine hydrochloride to give the oxime product ofStep C. The oxime product of Scheme 1, Step C can be converted to thebicyclic 4,5-dihydroisoxazole product of Step D in a 3+2 cyclization byseveral methods such as using an aqueous solution of sodium hypochloriteor an alternative oxidant such as N-chlorosuccinimide and in a solventsuch as tert-butyl methyl ether, toluene, dichloromethane, or xylene ata temperature of about 10-15° C. or with heating. The 2-fluoro, 5-bromophenyl group can be added to the dihydroisoxazole by generating theorganometallic reagent. The organometallic reagent can be generated from4-bromo-1-fluoro-2-iodo-benzene using halogen-metal exchange withreagents such as n-butyllithium or isopropylmagnesium chloride lithiumchloride complex and dropwise addition at a temperature range of about−78° C. to 15° C. in a solvent such as THF. A Lewis acid such as borontrifluoride diethyl etherate is then added to give the product of Scheme1, Step E. The resulting bicyclic tetrahydroisoxazole can be treatedwith zinc in acetic acid to form the ring opened product of Scheme 1,Step F. An alternate method to open the isoxazole ring uses Raney Nickelin a polar solvent such as ethanol under pressure with hydrogenationconditions. The product of Step F can then be reacted with benzoylisothiocyanate in a solvent such as dichloromethane or THF at atemperature of about 5° C. to room temperature to give the thioureacompound of Step G. The thiazine ring can be formed usingtrifluoromethanesulfonic anhydride and an organic base such as pyridinein a solvent such as dichloromethane at a temperature of about −20° C.to give the product of Step H. The hydroxymethyl protecting group suchas a trityl group can be removed in Scheme 1, Step I using methods wellknown in the art such as formic acid at room temperature or withp-toluenesulfonic acid monohydrate in solvents such as dichloromethaneand methanol to give the compound of Step I. The hydroxy methyl can beoxidized to the carboxylic acid using oxidizing agents such as2-iodoxybenzoic acid (IBX) at temperatures of 0-22° C. in a solvent suchas DMSO or addition of (diacetoxyiodo)benzene portionwise or all at oncein a solvent such as acetonitrile or acetonitrile and water withstirring at a temperature of about 5-25° C. to give the compound ofScheme 1, Step J. TEMPO can also be used as a catalyst in the oxidation.The Weinreb amide is prepared in Scheme 1, Step K from the acid productof Step J with the addition of N,O-dimethylhydroxylamine hydrochloride,an organic base, such as triethylamine, and a coupling reagent such asHATU. The mixture is stirred at room temperature to give the product ofStep K. Other coupling agents that could be used include CDI,carbodiimides such as DCC, DIC, or EDCI or other uronium or phosphoniumsalts of non-nucleophilic anions, such as HBTU, PyBOP, and PyBrOP. TheWeinreb amide is then converted to the ketone using an organometallicreagent such as a Grignard reagent or an organolithium reagent in Step Lin a solvent such as THF. The appropriate Grignard reagent can be addedas a solution in solvents such as ether or 2-methyltetrahydrofuran tothe Weinreb amide at a temperature of about −78° C. to 0° C. to give theketone of Step L. In Scheme 1, Step M, the R and ketone group of thecompound of Step L can be converted to a difluoro-R group usingDeoxo-Fluor® in a solvent such as dichloromethane at about −78° C. toroom temperature. Another alternative procedure involves pre-mixing thefluorinating reagent such as Deoxo-Fluor® with boron trifluoride-diethyletherate followed by the addition of the product of Scheme 1, Step L andtriethylamine trihydrofluoride to give the product of Scheme 1, Step M.Alternatively, other fluorinating agents that may be used which are wellknown in the art are, diethylaminosulfur trifluoride (also referred toas “DAST”), XtalFluor-E® or XtalFluor-M® with an additive such astriethylamine trihydrofluoride or FLUOLEAD™ using an additive such asHF-pyridine. The 5-bromo of the phenyl is converted to the amine using(1R,2R)—N,N′-dimethyl-1,2-cyclohexanediamine ortrans-N,N′dimethylcyclohexane-1,2-diamine in a solvent such as ethanoland adding sodium azide followed by sodium L-ascorbate and cupricsulfate. The reaction is heated to about 80-100° C. for several hoursand then worked up with an extraction using a solvent such as ethylacetate. The intermediate is then reduced under hydrogenation conditionsusing palladium on carbon such as 5-10% palladium in solvents such asmethanol or ethanol and THF at a pressure of about 40-50 psi of hydrogento give the aniline product of Scheme 1, Step N.

Alternatively in Scheme 2, the protected product of Scheme 1, Step A,can be treated with 4-(2-chloroacetyl)morpholino and a base such astetrabutyl ammonium hydrogen sulfate in a solvent such as toluene at atemperature of about 5° C. to give the product of Scheme 2, Step A. Themorpholino group can then serve as a leaving group in Scheme 2, Step B.For example, the product of Scheme 2, Step A can be treated with theappropriate Grignard reagent which can be prepared in situ fromisopropyl magnesium chloride lithium chloride complex and4-bromo-1-fluoro-2-iodobenzene or if the appropriate Grignard reagent isavailable, the reagent can be added directly to the product of Scheme 2,Step A at a temperature of about 5° C. to give the product of Scheme 2,Step B. The carbonyl acetate can be converted to an oxime withhydroxylamine hydrochloride and sodium acetate with heating to about 50°C. to give the product of Scheme 2, Step C. The oxime product of Scheme2, Step C can then be converted to the product of Scheme 2, Step D (thesame product as Scheme 1, Step E) using hydroquinone in a solvent suchas toluene and heating to reflux. The amine product of Scheme 2, Step Dcan be acylated with acetyl chloride using an organic base such as DMAPand pyridine in a solvent such as dichloromethane at a temperature ofabout 0-5° C. to give the product of Scheme 2, Step E. The product ofScheme 2, Step E can then be converted to the product of Scheme 3, StepA as discussed below.

In an alternate route, as described in Scheme 3, the isoxazole nitrogenof the compound of Scheme 1, Step E, is protected with an acetyl groupand the protecting group of the hydroxy methyl is removed in a two-stepprocedure. For example, the tetrahydroisoxazole is treated with anorganic base such as DMAP and pyridine in a solvent such asdichloromethane and acetyl chloride is added. The temperature ismaintained below about 10° C. and then allowed to stir at about roomtemperature. The reaction is diluted with water and extracted with asolvent such as dichloromethane. The organic extracts are washed with anaqueous acid such as 1 N hydrochloric acid and the aqueous extractedagain with a solvent such as dichloromethane followed by an aqueouswash. The organic solvent can be partially removed and an acid such asformic acid or p-toluenesulfonic acid monohydrate in solvents such asdichloromethane and methanol can added to deprotect the hydroxy methyl.The mixture can be stirred at room temperature or heated to atemperature of about 40° C. until deprotection of the hydroxy iscomplete to give the compound of Scheme 3, Step A. The hydroxy methylproduct of Scheme 3, Step A can be oxidized to the carboxylic acidproduct of Scheme 3, Step B in a manner analogous to the proceduredescribed in Scheme 1, Step J, and the Weinreb amide can be furtherprepared in a manner analogous to the procedure described in Scheme 1,Step K using a coupling agent such as CDI in a portionwise addition oradding at once with a solvent such as dichloromethane, cooling to −20°C. and stirring for about 1 hour and adding N,O-dimethylhydroxylaminehydrochloride portionwise or all at once. An organic base such astriethylamine can also be used to promote the reaction. Furtheradditions of CDI and N,O-dimethylhydroxylamine can be added untilcomplete reaction is observed to give the Weinreb amide product ofScheme 3, Step C. The ketone of Scheme 3, Step D can be formed from theWeinreb amide in a manner analogous to the procedure described in Scheme1, Step L. The ketone of Step D can be converted to a difluoro-R groupin a manner analogous to the procedure described in Scheme 1, Step M togive the product of Scheme 3, Step E. The acetyl tetrahydroisoxazole candeprotected under acidic conditions well known in the art such as usinghydrochloric acid and heating to about 100° C. to give the product ofScheme 3, Step F. The bicyclic tetrahydroisoxazole can be treated withzinc in acetic acid to form the ring opened product of Scheme 3, Step Gin a manner analogous to the procedure described in Scheme 1, Step F.The thiazine product of Scheme 3, Step H can be prepared in a one pot 2step reaction using benzoyl isothiocyanate in a manner analogous to theprocedure described in Scheme 1, Step G. The mixture is evaporated to aresidue and cyclohexane is added. The mixture is heated to about 60° C.and methyl tert-butyl ether is added to dissolve the residue. Thesolution is filtered and concentrated to dryness. The thiazine ring canthen be formed in a manner analogous to the procedure described inScheme 1, Step H to give the product of Scheme 3, Step H.

In Scheme 4, Step A, the aniline product of Scheme 1, Step N can becoupled with a heteroaromatic carboxylic acid utilizing couplingconditions well known in the art. One skilled in the art will recognizethat there are a number of methods and reagents for amide formationresulting from the reaction of carboxylic acids and amines. For example,the reaction of an appropriate aniline with an appropriate acid in thepresence of a coupling reagent and an amine base such asdiisopropylethylamine or triethylamine, will give a compound of Scheme4, Step A. Coupling reagents include carbodiimides such as DCC, DIC,EDCI, and aromatic oximes such as HOBt and HOAt. Additionally, uroniumor phosphonium salts of non-nucleophilic anions such as HBTU, HATU,PyBOP, and PyBrOP or a cyclic phosphoric anhydride such as T3P® can beused in place of the more traditional coupling reagents. Additives suchas DMAP may be used to enhance the reaction. Alternatively, the anilineamine can be acylated using substituted benzoyl chlorides in thepresence of a base such as triethylamine or pyridine. In Scheme 4, StepB, the protected thiazine amine can then be deprotected with an organicbase such as pyridine and O-methylhydroxylamine hydrochloride insolvents such as THF and ethanol and an organic base such as pyridine toprovide the compound of Formula II. Alternatively an inorganic base suchas lithium hydroxide in methanol may be used to deprotect the thiazineto provide the compound of Formula II.

Alternatively, in Scheme 5, the bromide product of Scheme 1, Step M isconverted to a protected aniline using trifluoroacetamide, copperiodide, a diamine such as trans, racemic-N,N′-dimethyl-1,2-cyclohexanediamine, an inorganic base such as potassium carbonate, and sodiumiodide with heating to about 100-130° C. to give the protected anilineproduct of Scheme 5, Step A. The protected aniline and thiazine aminecan then be deprotected stepwise. The trifluoroacetamide can behydrolyzed using a base such as 7 N ammonia in methanol to give ananiline and protected thiazine, the same product of Scheme 1, Step N.The thiazine can then be deprotected under conditions well known in theart and described in Scheme 4, Step B using O-methylhydroxylaminehydrochloride in a solvent such as ethanol and THF with an organic basesuch as pyridine followed by heating to about 55° C. or stirring at roomtemperature followed by concentration and purification to give theproduct of Scheme 5, Step B. Alternatively, the order of deprotectioncould be reversed with the thiazine deprotected first and the anilinedeprotected last. In Scheme 5, Step C, the aniline product of Step B canthen be reacted with the appropriate carboxylic acid or acid chloride asdescribed in Scheme 4, Step A to give the products of Formula II.

A pharmaceutically acceptable salt of the compounds of the invention,such as a hydrochloride salt, can be formed, for example, by reaction ofan appropriate free base of Formulas I, Ia, II, or IIa and anappropriate pharmaceutically acceptable acid such as hydrochloric acid,p-toluenesulfonic acid, or malonic acid in a suitable solvent such asdiethyl ether under standard conditions well known in the art.Additionally, the formation of such salts can occur simultaneously upondeprotection of a nitrogen protecting group. The formation of such saltsis well known and appreciated in the art. See, for example, Gould, P.L., “Salt selection for basic drugs,” International Journal ofPharmaceutics, 33: 201-217 (1986); Bastin, R. J., et al. “Salt Selectionand Optimization Procedures for Pharmaceutical New Chemical Entities,”

The following preparations and examples further illustrate theinvention.

Preparation 1 (2S)-1-Trityloxybut-3-en-2-ol

Scheme 1, step A: Stir trimethylsulfonium iodide (193.5 g, 948.2 mmol)in THF (1264 mL) at ambient temperature for 75 minutes. Cool mixture to−50° C. and add n-butyllithium (2.5 mol/L in hexanes, 379 mL, 948.2mmol) via cannula, over a period of 30 minutes. Allow the reaction togradually warm to −30° C. and stir for 60 minutes. Add(2S)-2-trityloxymethyl oxirane (100 g, 316.1 mmol) portion wise, keepingthe temperature below −10° C. After the complete addition, allow thereaction mixture to warm to room temperature and stir for 2 hours. Pourthe reaction into saturated ammonium chloride, separate the phases, andextract the aqueous phase with ethyl acetate. Combine the organic layersand dry over magnesium sulfate. Filter and concentrate under reducedpressure to give a residue. Purify the residue by silica gelchromatography, eluting with methyl t-butyl ether:hexanes (10-15%gradient), to give the title compound (56.22 g, 54%). ES/MS m/z 353(M+Na).

Alternate Preparation 1 (2S)-1-Trityloxybut-3-en-2-ol

Scheme 2, step A starting material: Add triphenylmethyl chloride (287 g,947.1 mmol), DMAP (7.71 g, 63.1 mmol) and triethylamine (140 g, 1383.5mmol) to a solution of (2S)-but-2-ene-1,2-diol (prepared as in JACS,1999, 121, 8649) (64.5 g, 631 mmol) in dichloromethane (850 mL). Stirfor 24 hours at 24° C. Add 1 N aqueous citric acid (425 mL). Separatethe layers and concentrate the organic extract under reduced pressure todryness. Add methanol (900 mL) and cool to 5° C. for 1 hour. Collect thesolids by filtration and wash with 5° C. methanol (50 mL). Discard thesolids and concentrate the mother liquor under reduced pressure todryness. Add toluene (800 mL) and concentrate to a mass of 268 g toobtain the title compound (129 g, 67%) in a 48 wt % solution of toluene.

Preparation 2 1-Morpholino-2-[(1S)-1-(trityloxymethyl)allyloxy]ethanone

Scheme 2, step A: Add tetrabutyl ammonium hydrogen sulfate (83.2 g,245.0 mmol) and 4-(2-chloroacetyl)morpholine (638.50 g, 3902.7 mmol) toa solution of 1-trityloxybut-3-en-2-ol (832.4, 2519 mmol) in toluene(5800 mL) that is between 0 and 5° C. Add sodium hydroxide (1008.0 g,25202 mmol) in water (1041 mL). Stir for 19 hours between 0 and 5° C.Add water (2500 mL) and toluene (2500 mL). Separate the layers and washthe organic extract with water (2×3500 mL). Concentrate the organicextract under reduced pressure to dryness. Add toluene (2500 mL) to theresidue and then add n-heptane (7500 mL) slowly. Stir for 16 hours.Collect the resulting solids by filtration and wash with n-heptane (1200mL). Dry the solid under vacuum to obtain the title compound (1075.7 g,98%).

Preparation 31-(5-Bromo-2-fluoro-phenyl)-2-[(1S)-1-(trityloxymethyl)allyloxy]ethanone

Scheme 2, step B: Add a 1.3 M solution of isopropyl magnesium chloridelithium chloride complex (3079 mL, 2000 mmol) in THF to a solution of4-bromo-1-fluoro-2-iodobenze (673.2 g, 2237.5 mmol) in toluene (2500 mL)at a rate to maintain the reaction temperature below 5° C. Stir for 1hour. Add the resulting Grignard solution (5150 mL) to a solution of1-morpholino-2-[(1S)-1-(trityloxymethyl)allyloxy]ethanone (500 g, 1093mmol) in toluene (5000 mL) at a rate to maintain the reactiontemperature below 5° C. Stir for 3 hours maintaining the temperaturebelow 5° C. Add additional prepared Grignard solution (429 mL) and stirfor 1 hour. Add a 1 N aqueous citric acid solution (5000 mL) at a rateto maintain the temperature below 5° C. Separate the layers and wash theorganic extract with water (5000 mL). Concentrate the solution underreduced pressure to dryness. Add methanol (2000 mL) to the residue andconcentrate to give the title compound as a residue (793 g, 73.4%potency, 83%).

Preparation 41-(5-Bromo-2-fluoro-phenyl)-2-[(1S)-1-(trityloxymethyl)allyloxy]ethanoneoxime

Scheme 2, step C: Add hydroxylamine hydrochloride (98.3 g) to1-(5-bromo-2-fluoro-phenyl)-2-[(1S)-1-(trityloxymethyl)allyloxy]ethanone(450 g, 707 mmol) and sodium acetate (174 g) in methanol (3800 mL). Heatthe solution to 50° C. for 2 hours. Cool to 24° C. and concentrate. Addwater (1000 mL) and toluene (1500 mL) to the residue. Separate thelayers and extract the aqueous phase with toluene (500 mL). Combine theorganic extract and wash with water (2×400 mL). Concentrate the solutionunder reduced pressure to give the title compound as a residue (567 g,61.4% potency, 88%).

Preparation 5 tert-Butyl 2-[(1S)-1-(trityloxymethyl)allyloxy]acetate

Scheme 1, step B: Add (2S)-1-trityloxybut-3-en-2-ol (74.67 g, 226.0mmol) to a solution of tetra-N-butylammonium sulfate (13.26 g, 22.6mmol) in toluene (376 mL). Add sodium hydroxide (50% mass) in water (119mL) followed by tert-butyl-2-bromoacetate (110.20 g, 565.0 mmol). Stirreaction mixture for 18 hours at ambient temperature. Pour into water,separate the phases, and extract the aqueous phase with ethyl acetate.Combine the organic layers and dry over magnesium sulfate. Filter themixture and concentrate under reduced pressure to give the titlecompound (77.86 g, 77%). ES/MS m/z 467 (M+Na).

Preparation 6 (1E)-2-[(1S)-1-(Trityloxymethyl)allyloxy]acetaldehydeoxime

Scheme 1, step C: Cool a solution of tert-butyl2-[(1S)-1-(trityloxymethyl)allyloxy]acetate (77.66 g, 174.7 mmol) indichloromethane (582.2 mL) to −78° C. Add a solution ofdiisobutylaluminum hydride in hexanes (1 mol/L, 174.7 mL) dropwise overa period of 35 minutes and maintain the temperature below −70° C. Stirat −78° C. for 5 hours. Add hydrochloric acid in water (2 mol/L, 192.1mL) to the reaction mixture dropwise, keeping the temperature below −60°C. Allow the reaction to gradually warm to ambient temperature and stirfor 60 minutes. Separate the organic extract and wash with saturatedsodium bicarbonate. Dry the solution over magnesium sulfate, filter, andconcentrate under reduced pressure to give a residue. Dissolve theresidue in dichloromethane. Add sodium acetate (28.66 g, 349.3 mmol),followed by hydroxylamine hydrochloride (18.21 g, 262.0 mmol). Stir atambient temperature for 18 hours. Pour into water, separate the phases,and extract the aqueous phase with dichloromethane. Combine the organiclayers and dry over magnesium sulfate. Filter the mixture andconcentrate under reduced pressure to give the title compound (68.38 g,101%). ES/MS m/z 386 (M−H).

Preparation 7 (3 aR,4S)-4-(Trityloxymethyl)-3,3a,4,6-tetrahydrofuro[3,4-c]isoxazole

Scheme 1, step D: Cool a solution of(1E)-2-[(1S)-1-(trityloxymethyl)allyloxy]acetaldehyde oxime (55.57 g,143.4 mmol) in tert-butyl methyl ether (717 mL) to 5° C. Add sodiumhypochlorite (5% in water, 591 mL, 430.2 mmol) dropwise, keeping thetemperature below 10° C. Stir at 10° C. for 30 minutes. Allow thereaction to warm to 15° C. Stir at 15° C. for 18 hours. Dilute thereaction mixture with ethyl acetate and wash with saturated sodiumbicarbonate. Separated the phases, wash the organic phase with a 5%sodium hydrogen sulphite solution and brine. Dry the solution overmagnesium sulfate, filter, and concentrate under reduced pressure togive a residue. Purify the residue by silica gel chromatography, elutingwith 50% methyl tert-butyl ether/dichloromethane:hexanes (20-27%gradient), to give the title compound (35.84 g, 65%). ES/MS m/z 408(M+Na).

Preparation 8(3aR,4S,6aR)-6a-(5-Bromo-2-fluoro-phenyl)-4-(trityloxymethyl)-3,3a,4,6-tetrahydrofuro[3,4-c]isoxazole

Scheme 1, step E: Cool a solution of 4-bromo-1-fluoro-2-iodo-benzene(86.94 g, 288.9 mmol) in THF (144.5 mL) and toluene (1445 mL) to −78° C.Add n-butyllithium (2.5 M in hexanes, 120 mL, 288.9 mmol) dropwise,keeping the temperature below −70° C. Stir for 30 minutes at −78° C. Addboron trifluoride diethyl etherate (36.5 mL, 288.9 mmol) dropwise,keeping temperature below −70° C. Stir the solution for 30 minutes at−78° C. Add a solution of(3aR,4S)-4-(trityloxymethyl)-3,3a,4,6-tetrahydrofuro[3,4-c]isoxazole(55.69 g, 144.5 mmol) in THF (482 mL) dropwise to the reaction, over aperiod of 30 minutes, keeping temperature below −65° C. Stir at −78° C.for 90 minutes. Rapidly add saturated ammonium chloride, keepingtemperature below −60° C. Pour into brine, and extract the aqueous phasewith ethyl acetate. Combine the organic extract and dry over magnesiumsulfate. Filter and concentrate under reduced pressure to give aresidue. Purify the residue by silica gel chromatography, eluting with10-15% diethyl ether:hexanes (0-70% gradient), to give the titlecompound (36.52 g, 45%). ES/MS m/e (⁷⁹Br/⁸¹Br) 560/562 [M+H].

Alternate Preparation 8

Scheme 2, step D: Heat a solution of1-(5-bromo-2-fluoro-phenyl)-2-[(1S)-1-(trityloxymethyl)allyloxy]ethanoneoxime (458 g, 502 mmol) and hydroquinone (56.3 g 511 mmol) in toluene(4000 mL) to reflux under nitrogen for 27 hours. Cool the solution to24° C. and add aqueous sodium carbonate (800 mL). Separate the layersand extract the aqueous phase with toluene (300 mL). Combine the organicextract and wash with water (2×500 mL). Concentrate the solution underreduced pressure to give a residue. Add isopropyl alcohol (1500 mL) andheat to reflux. Cool to 24° C. and collect the solids by filtration. Drythe solid under vacuum to obtain the title compound (212 g, 75%).

Preparation 91-[(3aR,4S,6aS)-6a-(5-Bromo-2-fluoro-phenyl)-4-(trityloxymethyl)-3,3a,4,6-tetrahydrofuro[3,4-c]isoxazol-1-yl]ethanone

Scheme 2, step E: Add acetyl chloride (35.56 g, 503.9 mmol) to asolution of(3aR,4S,6aR)-6a-(5-bromo-2-fluoro-phenyl)-4-(trityloxymethyl)-3,3a,4,6-tetrahydrofuro[3,4-c]isoxazole(235.3 g, 420 mmol), DMAP (5.13 g, 42.0 mmol), and pyridine (66.45 g,840.1 mmol) in dichloromethane (720 mL) under nitrogen, maintaininginternal temperature below 5° C. Stir for 1 hour and then add water (300mL) and 1 M sulfuric acid (300 mL). Stir the mixture for 10 minutes andallow the layers to separate. Collect the organic extract and wash withsaturated sodium carbonate (500 mL) and water (500 mL). Dry the solutionover magnesium sulfate. Filter and concentrate under reduced pressure togive1-[(3aR,4S,6aS)-6a-(5-Bromo-2-fluoro-phenyl)-4-(trityloxymethyl)-3,3a,4,6-tetrahydrofuro[3,4-c]isoxazol-1-yl]ethanone(235 g, 93%) as a grey solid.

Preparation 101-[(3aR,4S,6aS)-6a-(5-Bromo-2-fluorophenyl)-4-(hydroxymethyl)tetrahydro-1H,3H-furo[3,4-c][1,2]oxazol-1-yl]ethanone

Scheme 3, step A: In a 20 L jacketed reactor add acetyl chloride (290mL, 4075 mmol) to a solution of(3aR,4S,6aR)-6a-(5-bromo-2-fluoro-phenyl)-4-(trityloxymethyl)-3,3a,4,6-tetrahydrofuro[3,4-c]isoxazole(1996 g, 3384 mmol), DMAP (56.0 g, 458 mmol), pyridine (500 mL, 6180mmol) in dichloromethane (10 L) under nitrogen maintaining internaltemperature below 10° C. After complete addition (1 hour) warm to 20° C.and stir overnight. If reaction is incomplete, add acetyl chloride,DMAP, pyridine, and dichloromethane until complete reaction is observed.Cool the reaction mixture to 0° C. and slowly add water (5 L), stir thereaction mixture at 10° C. for 30 minutes and allow the layers toseparate. Collect the organic extract and wash the aqueous withdichloromethane (1 L). Wash the combined organic extracts with 1 Naqueous hydrochloric acid (2×4 L), extract the aqueous withdichloromethane (2×1 L). Wash the combined organic extracts with water(4 L) and remove the solvent under reduced pressure give total volume ofapproximately 5 L. Add 90% formic acid (1800 mL) and stand at ambienttemperature for 3 days. Warm to 40° C. for 2 hours then remove thesolvent under reduced pressure. Dilute the residue with methanol (4 L)and slowly add saturated aqueous sodium carbonate (3 L). Add solidsodium carbonate (375 g) to adjust the pH to 8-9. Stir at 45° C. for 1hour then cool to ambient temperature. Remove the solids by filtration,washing with methanol (4×500 mL) then treat with 2 N aqueous sodiumhydroxide (100 mL) and stand at ambient temperature for 1 hour. Removethe solids by filtration, washing with methanol (2×100 mL). Evaporatethe solvent under reduced pressure and partition the residue betweenethyl acetate (5 L) and water (2 L). Extract the aqueous with ethylacetate (2 L) and wash the combined organic extracts with brine (2×1 L).Remove the solvent under reduced pressure, add methyl tert-butyl ether(2.5 L) and evaporate to dryness. Add methyl tert-butyl ether (4 L) andstir at 65° C. for 1 hour cool to ambient temperature and collect thesolids by filtration, washing with methyl tert-butyl ether (3×500 mL).Dry under vacuum to a beige solid. Heat this solid in toluene (7.5 L) to110° C. until fully dissolved, cool to 18° C. over 1 hour, and stir atthis temperature for 1 hour. Warm to 40° C. and when precipitate forms,cool to 18° C. once more. Stir for 45 minutes then collect solids byfiltration, washing with toluene (2×500 mL). Dry the solid under vacuumto obtain the title compound (443.1 g, 36%, 95% purity by LCMS).Evaporate the filtrate under vacuum to give a residue. Purify theresidue by silica gel flash chromatography, eluting with 20% to 100%ethyl acetate in isohexane. Slurry the product containing fractions inmethyl tert-butyl ether (2 L) at 60° C. for 30 minutes, cool to ambienttemperature, and collect the solids by filtration, washing with methyltert-butyl ether (2×200 mL). Dry the solids under vacuum to give thetitle compound as a beige crystalline solid (304 g, 24%, 88% purity byLCMS). Evaporate the filtrate under vacuum to a residue. Purify theresidue by silica gel flash chromatography, eluting with 20% to 100%ethyl acetate in isohexane to give the title compound (57.8 g, 5%, 88%purity by LCMS). ES/MS: m/z (⁷⁹Br/⁸¹Br) 360.0/362.0 [M+H].

Alternate Preparation 10

Scheme 3, step A: Add1-[(3aR,4S,6aS)-6a-(5-bromo-2-fluoro-phenyl)-4-(trityloxymethyl)-3,3a,4,6-tetrahydrofuro[3,4-c]isoxazol-1-yl]ethanone(69 g, 114.5 mmol) to a 15° C. solution of p-toluenesulfonic acidmonohydrate (2.2 g, 11.45 mmol), dichloromethane (280 mL) and methanol(700 mL). Stir for 18 hours and then remove the solvent under reducedpressure. Dilute the residue with dichloromethane (350 mL) and add 1 Maqueous sodium carbonate (140 mL) and water (140 mL). Separate thelayers and evaporate the organic layer under reduced pressure. Addtoluene (350 mL) to the residue and heat to reflux for 1 hour. Cool to10-15° C. at a rate of 10° C./hour. Collect the solids by filtration andwash with toluene (70 mL). Dry the solid under vacuum to obtain thetitle compound (30 g, 65%) as a grey solid.

Preparation 11(3aR,4S,6aS)-1-Acetyl-6a-(5-bromo-2-fluoro-phenyl)-3,3a,4,6-tetrahydrofuro[3,4-c]isoxazole-4-carboxylicacid

Scheme 3, step B: Add water (2 L) to a suspension of1-[(4S,6aS)-6a-(5-bromo-2-fluoro-phenyl)-4-(hydroxymethyl)-3,3a,4,6-tetrahydrofuro[3,4-c]isoxazol-1-yl]ethanone(804.9 g, 2177 mmol), TEMPO (40.0 g, 251 mmol) in acetonitrile (4.5 L)in a 20 L jacketed reactor and cool to an internal temperature of 5° C.Add (diacetoxyiodo)benzene (1693 g, 4993.43 mmol) portionwise over 30minutes. Control the exotherm using reactor cooling and then hold at 20°C. until LCMS shows complete reaction. Slowly add a suspension of sodiumbisulfite (70 g, 672.68 mmol) in water (300 mL) at ambient temperature,maintaining the internal temperature below 25° C. Stir for 30 minutesand then cool to 5° C. Add water (2 L), then slowly add 47 wt % aqueoussodium hydroxide (780 mL) over a period of 1 hour maintaining theinternal temperature below 10° C. Add ethyl acetate (2 L) and isohexane(5 L), stir vigorously and separate the layers. Extract the biphasicorganic layers with water (1 L) and wash the combined aqueous withmethyl tert-butyl ether (2.5 L). Cool the aqueous extracts to 5° C. andslowly add 37% hydrochloric acid (1.4 L) over 30 minutes maintaining theinternal temperature around 5° C. Add ethyl acetate (5 L), separate thelayers and wash the organic with brine (3×1 L). Extract the combinedaqueous extracts with ethyl acetate (2.5 L), wash the combined organicswith brine (1 L), then dry with sodium sulfate, and filter. Dilute theorganics with heptane (2.5 L) and evaporate to dryness under reducedpressure. Add methyl tert-butyl ether (1.5 L) and heptane (1.5 L) andevaporate to dryness. Add heptane (2.5 L) and evaporate to drynesstwice. Add heptane (500 mL) and methyl tert-butyl ether (500 mL) andstir at 40° C. for 30 minutes then collect the precipitate byfiltration, washing with heptane/methyl tert-butyl ether (1:1, 1 L) thenmethyl tert-butyl ether (3×300 mL) and air dry to give the titlecompound as a beige crystalline solid (779 g, 91%). ES/MS: m/z(⁷⁹Br/⁸¹Br) 374.0/376.0 [M+H].

[α]_(D) ²⁰=−19.0° (C=1.004, chloroform).

Alternate Preparation 11

Scheme 3, step B: Add water (150 mL) and acetonitrile (150 mL) to1-[(4S,6aS)-6a-(5-bromo-2-fluoro-phenyl)-4-(hydroxymethyl)-3,3a,4,6-tetrahydrofuro[3,4-c]isoxazol-1-yl]ethanone(30 g, 73.3 mmol), TEMPO (1.14 g, 7.30 mmol) and (diacetoxyiodo) benzene(51.9 g, 161 mmol). Cool to 15° C. and stir for 2 hours. Slowly addsodium thiosulfate (21 g) and potassium carbonate (22 g) in water (150mL) at ambient temperature. Stir for 1 hour and then add methyltert-butyl ether (150 mL). Separate the layers and adjust the pH of theaqueous layer to 2-3 with concentrated sulfuric acid. Add ethyl acetate(150 mL) and separate the layers. Evaporate the organic layer to drynessunder reduced pressure. Add n-heptane (90 mL) and heat to reflux for 1hour. Cool to 15° C. and then collect the precipitate by filtration,washing with n-heptane (90 mL). Dry under vacuum to give the titlecompound as a white solid (27 g, 98%).

Preparation 12(3aR,4S,6aS)-1-Acetyl-6a-(5-bromo-2-fluorophenyl)-N-methoxy-N-methyltetrahydro-1H,3H-furo[3,4-c][1,2]oxazole-4-carboxamide

Scheme 3, step C: In a 10 L jacketed reactor, cool a solution of(3aR,4S,6aS)-1-acetyl-6a-(5-bromo-2-fluoro-phenyl)-3,3a,4,6-tetrahydrofuro[3,4-c]isoxazole-4-carboxylicacid (771 g, 2019 mmol) in dichloromethane (7.0 L) to 0° C. undernitrogen and add CDI (400 g, 2421 mmol) portionwise over 40 minutes.Cool the reactor jacket to −20° C. and stir for 1 hour and then addN,O-dimethylhydroxylamine hydrochloride (260.0 g, 2612 mmol) portionwiseover about 30 minutes. Stir at −20° C. for 1 hour, at 0° C. for 2 hours,and at 10° C. for 7 hours. Add CDI (175 g, 1058 mmol) and stir at 10° C.overnight. Add further CDI (180 g, 1088 mmol) at 10° C. and stir for 1hour then add N,O-dimethylhydroxylamine hydrochloride (140 g, 1407 mmol)and continue stirring at 10° C. If the reaction is incomplete, furthercharges of CDI followed by N,O-dimethylhydroxylamine hydrochloride canbe made until complete reaction is observed. Cool the reaction mixtureto 5° C. and wash with 1 N aqueous hydrochloric acid (5 L) then 2 Naqueous hydrochloric acid (5 L). Extract the combined aqueous solutionwith dichloromethane (1 L), combine the organic extract and wash withwater (2.5 L), 1 N aqueous sodium hydroxide (2.5 L), and water (2.5 L),dry over magnesium sulfate, filter, and evaporate under reduced pressureto give a residue. Add methyl tert-butyl ether (3 L) and evaporate underreduced pressure. Add further methyl tert-butyl ether (2 L) and stir at50° C. for 1 hour, cool to 25° C. and stir for 30 minutes. Collect theresulting solids by filtration, wash with methyl tert-butyl ether (2×500mL) and dry under vacuum to give the title compound (760 g, 88%) as awhite solid. ES/MS: m/z (⁷⁹Br/⁸¹Br) 417.0/419.0 [M+H].

Alternate Preparation 12

Scheme 3, step C: Cool a solution of(3aR,4S,6aS)-1-acetyl-6a-(5-bromo-2-fluoro-phenyl)-3,3a,4,6-tetrahydrofuro[3,4-c]isoxazole-4-carboxylicacid (27 g, 70.7 mmol) in N,N-dimethylformamide (135 mL) to 0° C. undernitrogen and add CDI (14.9 g, 91.9 mmol). Stir for 1 hour and then addN,O-dimethylhydroxylamine hydrochloride (9.0 g, 92 mmol) andtriethylamine (14.3 g, 141 mmol). Stir at 15° C. for 16 hours. Cool thereaction mixture to 0° C. and add 0.5 M aqueous sulfuric acid (675 mL).Stir for 1 hour. Collect the resulting solids by filtration. Slurry thesolids in methyl tert-butyl ether (90 mL) for 1 hour. Collect the solidsby filtration, wash with methyl tert-butyl ether (30 mL). Dry undervacuum to give the title compound (23 g, 78%) as a solid.

Preparation 131-[(3aR,4S,6aS)-1-Acetyl-6a-(5-bromo-2-fluoro-phenyl)-3,3a,4,6-tetrahydrofuro[3,4-c]isoxazol-4-yl]ethanone

Scheme 3, step D: In a 20 L jacketed reactor, cool a solution of(3aR,4S,6aS)-1-acetyl-6a-(5-bromo-2-fluorophenyl)-N-methoxy-N-methyltetrahydro-1H,3H-furo[3,4-c][1,2]oxazole-4-carboxamide(654.0 g, 1536 mmol) in THF (10 L) to −60° C. and add a 3.2 M solutionof methylmagnesium bromide in 2-methyltetrahydrofuran (660 mL, 2110mmol) dropwise, while maintaining the internal temperature below −40° C.Stir the reaction mixture at −40° C. for 30 minutes then cool to −50° C.and add a solution of 1 N aqueous hydrochloric acid (2 L) in THF (2 L)maintaining the internal temperature below −38° C. Increase thetemperature to 10° C. and add ethyl acetate (5 L) and water (1 L), stirand allow internal temperature to reach 5° C. and separate the layers.Extract the aqueous layer with ethyl acetate (1 L) and combine theorganic extracts. Wash the organic extracts with water (2 L) and extractthe aqueous layer with ethyl acetate (1 L). Combine the organic extractand wash with brine (3×2 L) then dry over magnesium sulfate, filter, andevaporate under reduced pressure to a residue. Add cyclohexane (2.5 L),stir at 60° C. for 1 hour then at 20° C. for 30 minutes, and collect thesolid by filtration, washing with cyclohexane (500 mL). Dry the solidunder vacuum to obtain the title compound as a white solid (565 g, 99%).ES/MS: m/z (⁷⁹Br/⁸¹Br) 372.0/374.0 [M+H], [α]_(D) ²⁰=−58.0° (C=1.000,chloroform).

Alternate Preparation 13

Scheme 3, step D: Cool a solution of(3aR,4S,6aS)-1-acetyl-6a-(5-bromo-2-fluorophenyl)-N-methoxy-N-methyltetrahydro-1H,3H-furo[3,4-c][1,2]oxazole-4-carboxamide(4.0 g, 9.59 mmol) in THF (60 mL) to −5° C. and add a 3.0 M solution ofmethylmagnesium bromide in 2-methyltetrahydrofuran (5.0 mL, 15 mmol)dropwise, while maintaining the internal temperature between −5 and 0°C. Stir the reaction mixture between −5 and 0° C. for 60 minutes thenadd a solution of saturated ammonium chloride (20 mL). Add methyltert-butyl ether (40 mL), allow the internal temperature to reach 5° C.and separate the layers. Evaporate the organic layer under reducedpressure to a residue. Add n-heptane (50 mL), stir, and collect thesolid by filtration. Dry the solid under vacuum to obtain the titlecompound as a solid (3.0 g, 77%).

Preparation 141-[(3aR,4S,6aS)-6a-(5-Bromo-2-fluorophenyl)-4-(1,1-difluoroethyl)tetrahydro-1H,3H-furo[3,4-c][1,2]oxazol-1-yl]ethanone

Scheme 3, step E: Add1-[(3aR,4S,6aS)-1-acetyl-6a-(5-bromo-2-fluoro-phenyl)-3,3a,4,6-tetrahydrofuro[3,4-c]isoxazol-4-yl]ethanone(5.08 g, 13.6 mmol) in a single portion to a stirred suspension ofXtalFluor-M® (10.02 g, 39.18 mmol) in anhydrous dichloromethane (100 mL)at 0-5° C. Stir the mixture for 10 minutes and add triethylaminetrihydrofluoride (4.5 mL, 27 mmol) dropwise over 10 minutes. Stir thereaction mixture in the ice-bath for 8 hours then warm to ambienttemperature and stir overnight. Add saturated aqueous sodium carbonate(100 mL) and stir for 1 hour. Separate the layers and extract theaqueous with dichloromethane (2×50 mL). Combine the organic extracts andwash with saturated aqueous sodium bicarbonate (100 mL), 2 N aqueoushydrochloric acid (2×100 mL), and brine (100 mL). Evaporate to drynessto a light brown solid and dissolve in methyl tert-butyl ether (300 mL)at 60° C. Filter the hot solution and evaporate the filtrate to give abrown solid (5.3 g, 81%, 82% purity by LCMS) that is used withoutfurther purification. ES/MS: m/z (⁷⁹Br/⁸¹Br) 393.8/395.8 [M+H].

Alternate Preparation 14

Scheme 3, step E: Add XtalFluor-M® (1.21 kg, 4.73 mol) in portions to astirred solution of1-[(3aR,4S,6aS)-1-acetyl-6a-(5-bromo-2-fluoro-phenyl)-3,3a,4,6-tetrahydrofuro[3,4-c]isoxazol-4-yl]ethanone(565 g, 1.51 mol) in anhydrous dichloromethane (5 L) at −14° C. Stir themixture for 10 minutes and add triethylamine trihydrofluoride (550 g,3.34 mol) dropwise over 20 minutes. Stir the reaction mixture at −10° C.for approximately 10 hours then warm to ambient temperature and stirovernight. Add 50% aqueous sodium hydroxide (750 mL) slowly, maintainingthe internal temperature below 10° C., then add water (1.5 L) andsaturated aqueous sodium hydrogen carbonate (1 L) and stir for 30minutes. Separate the layers and extract the aqueous withdichloromethane (1 L). Combine the organic extracts and wash with brine(3 L), 2 N aqueous hydrochloric acid (5 L), and brine (3 L). Evaporateto give a residue and purify by silica gel chromatography eluting with50-100% dichloromethane in iso-hexane then 10% methyl tert-butyl etherin dichloromethane to give the title compound as a white powder (467 g,73%, 94% purity by LCMS). ES/MS: m/z (⁷⁹Br/⁸¹Br) 393.8/395.8 [M+H].

Preparation 15(3aR,4S,6aS)-6a-(5-Bromo-2-fluoro-phenyl)-4-(1,1-difluoroethyl)-3,3a,4,6-tetrahydro-1H-furo[3,4-c]isoxazole

Scheme 3, step F: Add 37 wt % aqueous hydrochloric acid (1.3 L, 16 mol)to a solution of1-[(3aR,4S,6aS)-6a-(5-bromo-2-fluorophenyl)-4-(1,1-difluoroethyl)tetrahydro-1H,3H-furo[3,4-c][1,2]oxazol-1-yl]ethanone(570 g, 1.45 mol) in 1,4-dioxane (5 L) in a 10 L jacketed reactor andstir at 100° C. for approximately 3 hours or until LCMS shows completereaction. Cool the reaction mixture to 10° C., dilute with water (1 L)and add a mixture 50 wt % aqueous sodium hydroxide solution (800 mL) andwater (1 L) slowly, maintaining the internal temperature below 20° C.Add ethyl acetate (2.5 L) and stir vigorously, before separating thelayers and washing the organic phase with brine (2 L), further brine (1L), and water (1 L). Dry over magnesium sulfate, filter, and concentrateto dryness under reduced pressure to give a residue. Add cyclohexane(2.5 L) and evaporate to dryness then repeat to obtain the titlecompound as a brown oil (527 g, 89%, 86% purity by LCMS). ES/MS: m/z(⁷⁹Br/⁸¹Br) 351.8/353.8 [M+H].

Preparation 16[(2S,3R,4S)-4-Amino-4-(5-bromo-2-fluorophenyl)-2-(1,1-difluoroethyl)tetrahydrofuran-3-yl]methanol

Scheme 3, step G: Add zinc powder (6.0 g, 92 mmol) to a solution of(3aR,4S,6aS)-6a-(5-bromo-2-fluoro-phenyl)-4-(1,1-difluoroethyl)-3,3a,4,6-tetrahydro-1H-furo[3,4-c]isoxazole(5.06 g, 13.4 mmol) in acetic acid (100 mL) at ambient temperature andstir overnight. Dilute the mixture with ethyl acetate (200 mL) and water(300 mL) and stir vigorously while adding sodium carbonate (97 g, 915mmol). Separate the layers and wash the organic layer with brine (2×200mL), dry over magnesium sulfate, filter, and concentrate to give aresidue. Purify the residue by silica gel chromatography eluting with 0%to 100% methyl tert-butyl ether in isohexane to give the title compoundas a waxy solid (4.67 g, 89%, 90% purity by LCMS). ES/MS: m/z(⁷⁹Br/⁸¹Br) 354.0/356.0 [M+H].

Alternate Preparation 16

Scheme 3, step G: Add zinc powder (200 g, 3.06 mol) portionwise to asolution of(3aR,4S,6aS)-6a-(5-bromo-2-fluoro-phenyl)-4-(1,1-difluoroethyl)-3,3a,4,6-tetrahydro-1H-furo[3,4-c]isoxazole(304 g, 75% purity, 647 mmol) in acetic acid (2 L) and water (2 L) at20° C. then warm to 40° C. and stir overnight. Dilute the mixture water(2 L) and stir vigorously while adding sodium carbonate (4 kg, 43.4 mol)then adjust to pH 8-9 with further sodium carbonate. Add ethyl acetate(5 L) and water (2.5 L), stir for 30 minutes and filter throughdiatomaceous earth washing with 2:1 acetonitrile/water. Separate thelayers, extract the aqueous with ethyl acetate (2×2.5 L) and wash thecombined organic extracts with brine (2×2.5 L), dry over magnesiumsulfate, filter, and concentrate to give a residue. Purify the residueby SFC, column. Chiralpak AD-H (5), 50×250 mm; eluent: 12% ethanol (0.2%diethylmethylamine in CO₂; flow rate: 340 g/minute at UV 220 nm to givethe title compound as a white solid (197.7 g, 84%). [α]_(D) ²⁰=−6.93°(C=0.678, chloroform). ES/MS: m/z (⁷⁹Br/⁸¹Br) 354.0/356.0 [M+H].

Preparation 17[(2S,3R,4S)-4-Amino-4-(5-bromo-2-fluoro-phenyl)-2-(trityloxymethyl)tetrahydrofuran-3-yl]methanol

Scheme 1, step F: Add(3aR,4S,6aR)-6a-(5-bromo-2-fluoro-phenyl)-4-(trityloxymethyl)-3,3a,4,6-tetrahydrofuro[3,4-c]isoxazole(31.30 g, 55.9 mmol) to acetic acid (186 mL) to give a suspension. Addzinc (25.6 g, 391 mmol) and stir the reaction mixture vigorously for 18hours. Dilute the mixture with toluene and filter through diatomaceousearth. Concentrate the filtrate under reduced pressure. Solubilize theresidue with ethyl acetate, wash with brine, and saturated sodiumbicarbonate. Separate the phases, dry over magnesium sulfate, filter,and concentrate under reduced pressure to give the title compound (31.35g, 99%). ES/MS m/e (⁷⁹Br/⁸¹Br) 562/564 [M+H].

Preparation 18N-[[(3S,4R,5S)-3-(5-Bromo-2-fluoro-phenyl)-4-(hydroxymethyl)-5-(trityloxymethyl)tetrahydrofuran-3-yl]carbamothioyl]benzamide

Scheme 1, step G: Dissolve[(2S,3R,4S)-4-amino-4-(5-bromo-2-fluoro-phenyl)-2-(trityloxymethyl)tetrahydrofuran-3-yl]methanol(31.35 g, 55.73 mmol) in dichloromethane (557 mL) and cool to 5° C. Addbenzoyl isothiocyanate (9.74 mL, 72.45 mmol). After addition iscomplete, allow the reaction mixture to warm to room temperature andstir for 2 hours. Pour into saturated sodium bicarbonate, separate thephases, and extract the aqueous phase with dichloromethane. Combine theorganic extract and dry over magnesium sulfate. Filter the solution andconcentrate under reduced pressure to give the title compound (42.95 g,106%). ES/MS m/e (⁷⁹Br/⁸¹Br) 747/749 [M+Na].

Preparation 19N-[(4aS,5S,7aS)-7a-(5-Bromo-2-fluorophenyl)-5-(1,1-difluoroethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-c][1,3]thiazin-2-yl]benzamide

Scheme 3, step H: Add benzoyl isothiocyanate (1.80 mL, 13.3 mmol,) to asolution of[(2S,3R,4S)-4-amino-4-(5-bromo-2-fluorophenyl)-2-(1,1-difluoroethyl)tetrahydrofuran-3-yl]methanol(4.67 g, 11.9 mmol) in dichloromethane (20 mL) at ambient temperaturefor 1 hour until LCMS shows reaction is complete. Evaporate the reactionmixture to a residue under vacuum. Add cyclohexane (50 mL), warm to 60°C. and add methyl tert-butyl ether until precipitate is fully dissolved(100 mL). Filter the hot solution, cool to room temperature and slowlyevaporate under reduced pressure until formation of a white precipitate.Remove the solvent under reduced pressure and dissolve the residue inanhydrous dichloromethane (30 mL), add pyridine (2.4 mL, 30 mmol), andcool the solution to −25° C. Add trifluoromethanesulfonic anhydride (2.2mL 13 mmol) dropwise over 30 minutes and allow to warm 0° C. over 1hour. Wash the reaction mixture with water (25 mL), 2 N aqueoushydrochloric acid (25 mL), water (25 mL), aqueous saturated sodiumbicarbonate (25 mL), and water (25 mL), dry over magnesium sulfate,filter, and concentrated to dryness. Purify the residue by silica gelchromatography eluting with 5% methyl tert-butyl ether indichloromethane to give the title compound as a light yellow foam (5.0g, 76%, 90% purity by LCMS). ES/MS: m/z (⁷⁹Br/⁸¹Br) 499.0/501.0 [M+H].

Alternate Preparation 19

Scheme 3, step H: Add benzoyl isothiocyanate (98 mL, 724.9 mmol,) to asolution of[(2S,3R,4S)-4-amino-4-(5-bromo-2-fluorophenyl)-2-(1,1-difluoroethyl)tetrahydrofuran-3-yl]methanol(197.6 g, 546.7 mmol) in dichloromethane (1.2 L) at 30° C. for 1 hour.Add CDI (101 g, 610.4 mmol) and stir at ambient temperature for 3 hours.Further charges of CDI can be made to ensure complete consumption of thethiourea intermediate. Heat to 90° C. for 42 hours and cool the solutionto ambient temperature. Dilute the reaction mixture with ethyl acetate(2 L) and add 2 N aqueous hydrochloric acid (2 L), stir, add brine (1 L)and separate the layers. Wash the organic layer with 2 N aqueoushydrochloric acid (0.5 L), brine (2×1 L) and aqueous saturated sodiumbicarbonate (1 L). Dry over magnesium sulfate, filter, and concentrateto give a residue. Purify the residue by silica gel chromatographyeluting with 0-100% ethyl acetate in iso-hexane to give the titlecompound as a light yellow solid (234 g, 83%). ES/MS: m/z (⁷⁹Br/⁸¹Br)499.0/501.0 [M+H].

Preparation 20N-[(4aS,5S,7aS)-7a-(5-Bromo-2-fluoro-phenyl)-5-(trityloxymethyl)-4,4a,5,7-tetrahydrofuro[3,4-c][1,3]thiazin-2-yl]benzamide

Scheme 1, step H: DissolveN-[[(3S,4R,5S)-3-(5-bromo-2-fluoro-phenyl)-4-(hydroxymethyl)-5-(trityloxymethyl)tetrahydrofuran-3-yl]carbamothioyl]benzamide(42.95 g, 59.18 mmol) in dichloromethane (591 mL) and cool to −20° C.Add pyridine (12.0 mL, 148.0 mmol), followed by trifluoromethanesulfonicanhydride (10.97 mL, 65.10 mmol). Monitor the addition keeping thetemperature below −20° C. Stir the reaction mixture at −20° C. for 30minutes. Allow the reaction mixture to warm to room temperature. Pourinto saturated ammonium chloride, separate the phases, and extract theaqueous phase with dichloromethane. Combine the organic extract and dryover magnesium sulfate. Filter the solution and concentrate underreduced pressure to give the title compound (45.24 g, 108%). ES/MS m/e(⁷⁹Br/⁸¹Br) 707/709 [M+H].

Preparation 21N-[(4aS,5S,7aS)-7a-(5-Bromo-2-fluoro-phenyl)-5-(hydroxymethyl)-4,4a,5,7-tetrahydrofuro[3,4-d][1,3]thiazin-2-yl]benzamide

Scheme 1, step I: DissolveN-[(4aS,5S,7aS)-7a-(5-bromo-2-fluoro-phenyl)-5-(trityloxymethyl)-4,4a,5,7-tetrahydrofuro[3,4-d][1,3]thiazin-2-yl]benzamide(45.24, 63.93 mmol) in formic acid (160 mL) and stir at ambienttemperature for 1 hour. Add water (29 mL) over a period of 5 minutes.Stir for 50 minutes. Concentrate the mixture under reduced pressure to aresidue. Dissolve the residue in methanol (639 mL), add triethylamine(26.7 mL, 191.8 mmol), and stir overnight at ambient temperature. Pourinto brine, separate the phases, and extract the aqueous phase withchloroform. Combine the organic extract and dry over magnesium sulfate.Filter and concentrate under reduced pressure to give a residue. Purifythe residue by silica gel chromatography, eluting with acetone:hexanes(25-38% gradient), to give the title compound (16.04 g, 54%). ES/MS m/e(⁷⁹Br/⁸¹Br) 465/467 [M+H].

Preparation 22(4aS,5S,7aS)-2-Benzamido-7a-(5-bromo-2-fluoro-phenyl)-4,4a,5,7-tetrahydrofuro[3,4-d][1,3]thiazine-5-carboxylicacid

Scheme 1, step J: AddN-[(4aS,5S,7aS)-7a-(5-bromo-2-fluoro-phenyl)-5-(hydroxymethyl)-4,4a,5,7-tetrahydrofuro[3,4-d][1,3]thiazin-2-yl]benzamide(16.04 g, 34.47 mmol) to DMSO (172 mL). Add 2-iodoxybenzoic acid (35.56g, 120.70 mmol) and stir at ambient temperature for 3 hours. Dilute thereaction mixture with chloroform (300 mL) and pour into saturatedammonium chloride (400 mL). Separate the organic phase and dry overmagnesium sulfate. Filter the solution and concentrate under reducedpressure to give a residue. Dissolve the residue in ethyl acetate (400mL) and wash with saturated ammonium chloride (2×250 mL). Separate theorganic phase, dry over magnesium sulfate, filter, and concentrate underreduced pressure to give a residue. Dissolve the residue in adichloromethane:methanol mixture and add diethyl ether until a solidprecipitates. Collect the solid by filtration and dry under reducedpressure to give the title compound (5.78 g, 35%). ES/MS m/e (⁷⁹Br/⁸¹Br)479/481 [M+H].

Preparation 23(4aS,5S,7aS)-2-Benzamido-7a-(5-bromo-2-fluoro-phenyl)-N-methoxy-N-methyl-4,4a,5,7-tetrahydrofuro[3,4-d][1,3]thiazine-5-carboxamide

Scheme 1, step K: Dissolve(4aS,5S,7aS)-2-benzamido-7a-(5-bromo-2-fluoro-phenyl)-4,4a,5,7-tetrahydrofuro[3,4-d][1,3]thiazine-5-carboxylicacid (5.78 g, 12.1 mmol) in dichloromethane (201 mL) andN,O-dimethylhydroxylamine hydrochloride (1.76 g, 18.1 mmol). Addtriethylamine (5.29 mL, 36.2 mmol) followed by HATU (7.02 g, 18.1 mmol).Stir at ambient temperature for 3 days. Pour into saturated ammoniumchloride, separate the phases, and extract the aqueous phase with ethylacetate. Combine the organic extracts and dry over magnesium sulfate.Filter and concentrate under reduced pressure to give a residue. Purifythe residue by silica gel chromatography, eluting with ethylacetate:dichloromethane (0-50% gradient) to give the title compound(4.15 g, 66%). ES/MS m/e (⁷⁹Br/⁸¹Br) 522/524 [M+H].

Preparation 24N-[(4aS,5S,7aS)-5-Acetyl-7a-(5-bromo-2-fluoro-phenyl)-4,4a,5,7-tetrahydrofuro[3,4-d][1,3]thiazin-2-yl]benzamide

Scheme 1, step L: Add dropwise to a −78° C. solution of(4aS,5S,7aS)-2-benzamido-7a-(5-bromo-2-fluoro-phenyl)-N-methoxy-N-methyl-4,4a,5,7-tetrahydrofuro[3,4-d][1,3]thiazine-5-carboxamide(1.51 g, 2.89 mmol) in THF (57.8 mL) methylmagnesium bromide (3.0 mol/Lin diethyl ether, 4.8 mL, 14.5 mmol). Stir the reaction at −78° C. for 5minutes and allow to gradually warm to ambient temperature. Stir for 30minutes. Quench the reaction with methanol (4 mL), dilute with saturatedammonium chloride, and extract with ethyl acetate. Combine the organicextract and dry over sodium sulfate. Filter and concentrate underreduced pressure to give a residue. Purify the residue by silica gelchromatography, eluting with ethyl acetate:hexanes (0-100% gradient) togive the title compound (1.28 g, 93%). ES/MS m/e (⁷⁹Br/⁸¹Br) 477/479[M+Na].

Preparation 24bN-[(5S,7aS)-7a-(5-Bromo-2-fluorophenyl)-5-propanoyl-4a,5,7,7A-tetrahydro-4H-furo[3,4-d][1,3]thiazin-2-yl]benzamide

Scheme 1, step L: Add(4aS,5S,7aS)-2-benzamido-7a-(5-bromo-2-fluoro-phenyl)-N-methoxy-N-methyl-4,4a,5,7-tetrahydrofuro[3,4-d][1,3]thiazine-5-carboxamide(3.00 g, 5.74 mmol) to THF (115 mL) under nitrogen and cool to −78° C.Add a solution of ethylmagnesium bromide (1.0 mol/L) in THF (28.7 mL,28.7 mmol) to the reaction dropwise, maintaining the temperature at −78°C. After 1 hour, add methanol (10 mL) in one portion and then pour ontosaturated ammonium chloride solution. Extract the mixture with ethylacetate, dry the organic extracts over magnesium sulphate, filter, andconcentrate in vacuo to give a residue. Purify the residue by silica gelchromatography, eluting with hexanes/ethyl acetate (100-40% gradient) togive the title compound (2.844 g, 5.788 mmol, 100%). ES/MS m/z(⁷⁹Br/⁸¹Br) 491/493/513/515 (M+H/Na).

Preparation 24cN-[(5S,7aS)-7a-(5-Bromo-2-fluorophenyl)-5-(cyclopropylcarbonyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-2-yl]benzamide

Scheme 1, step L: Add dropwise to a −78° C. solution of(4aS,5S,7aS)-2-benzamido-7a-(5-bromo-2-fluoro-phenyl)-N-methoxy-N-methyl-4,4a,5,7-tetrahydrofuro[3,4-d][1,3]thiazine-5-carboxamide(1.51 g, 2.89 mmol) in THF (51.2 mL) cyclopropylmagnesium bromide (1.0mol/L in 2-methyltetrahydrofuran, 14 mL, 14.4 mmol). Stir the reactionat −78° C. for 5 minutes and allow to gradually warm to ambienttemperature. Stir for 30 minutes. Quench the reaction with methanol (4mL), dilute with saturated ammonium chloride, and extract with ethylacetate. Combine the organic extract and dry over sodium sulfate. Filterand concentrate under reduced pressure to give a residue. Purify theresidue by silica gel chromatography, eluting with ethyl acetate:hexanes(0-100% gradient) to give the title compound (1.299 g, 89%). ES/MS m/z(⁷⁹Br/⁸¹Br) 503/505 [M+H].

Preparation 25N-[(4aS,5S,7aS)-7a-(5-Bromo-2-fluoro-phenyl)-5-(1,1-difluoroethyl)-4,4a,5,7-tetrahydrofuro[3,4-d][1,3]thiazin-2-yl]benzamide

Scheme 1, step M: Add together dichloromethane (34 mL), Deoxo-Fluor®(1.52 mL, 6.88 mmol), and boron trifluoride diethyl etherate (0.89 mL,6.88 mmol). Stir at ambient temperature for 2 hours. AddN-[(4aS,5S,7aS)-5-acetyl-7a-(5-bromo-2-fluoro-phenyl)-4,4a,5,7-tetrahydrofuro[3,4-d][1,3]thiazin-2-yl]benzamide(0.821 g, 1.72 mmol) in one portion, followed by triethylaminetrihydrofluoride (1.13 mL, 6.88 mmol). Stir at ambient temperature for18 hours. Pour into saturated ammonium chloride, separate the phases,and extract the aqueous phase with ethyl acetate. Combine the organicextract and dry over magnesium sulfate. Filter and concentrate underreduced pressure to give a residue. Purify the residue by silica gelchromatography, eluting with dichloromethane:hexanes (80-100% gradient),to give the title compound (0.552 g, 64%). ES/MS m/e (⁷⁹Br/⁸¹Br) 499/501[M+H].

Preparation 25bN-[(4aS,5S,7aS)-7a-(5-Bromo-2-fluorophenyl)-5-(1,1-difluoropropyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-2-yl]benzamide

Scheme 1, step M: AddN-[(5S,7aS)-7a-(5-bromo-2-fluorophenyl)-5-propanoyl-4a,5,7,7A-tetrahydro-4H-furo[3,4-d][1,3]thiazin-2-yl]benzamide(0.486 g, 0.989 mmol) to dichloromethane (19.8 mL) under nitrogen at−78° C., followed by Deoxo-Fluor® (1.75 g, 1.75 mL, 3.96 mmol). Stir for30 minutes at −78° C. and then warm to room temperature overnight. Pourthe reaction onto saturated sodium bicarbonate solution and extract withdichloromethane. Separate organics using a hydrophobic frit andconcentrate in vacuo to give a residue. Purify the residue by silica gelchromatography, eluting with hexanes/ethyl acetate (100-50% gradient) togive the crude title compound with an impurity (0.230 g, 0.448 mmol,45%) that is used without further purification. ES/MS m/z 513/515 (M+H).

Preparation 25cN-[(4aS,5S,7aS)-7a-(5-Bromo-2-fluoro-phenyl)-5-[cyclopropyl(difluoro)methyl]-4,4a,5,7-tetrahydrofuro[3,4-d][1,3]thiazin-2-yl]benzamide

Scheme 1, step M: Add Deoxo-Fluor® (1.9 mL, 6.68 mmol) to an ambientsolution of N-[(5S,7aS)-7a-(5-bromo-2-fluorophenyl)-5-(cyclopropylcarbonyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-2-yl]benzamide(0.812 g, 1.61 mmol) in dichloromethane (24 mL). Stir at ambienttemperature for 18 hours. Pour into saturated sodium bicarbonate andextract the aqueous phase with ethyl acetate (3×). Combine the organicextracts and dry over sodium sulfate. Filter and concentrate underreduced pressure to give a residue. Purify the residue by silica gelchromatography, eluting with ethyl acetate:hexanes (5-100% gradient), togive the title compound (41 mg, 5%). ES/MS m/z (⁷⁹Br/⁸¹Br) 525/527[M+H].

Preparation 26N-[(5S,7aS)-5-(1,1-Difluoroethyl)-7a-{2-fluoro-5-[(trifluoroacetyl)amino]phenyl}-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-2-yl]benzamide

Scheme 4, step A: DissolveN-[(4aS,5S,7aS)-7a-(5-bromo-2-fluorophenyl)-5-(1,1-difluoroethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-2-yl]benzamide(234 g, 454.6 mmol) in 1,4-dioxane (2 L) and add 4 Å molecular sieves(37 g), 2,2,2-trifluoroacetamide (91 g, 780.9 mmol), finely groundpotassium carbonate (114 g, 824.9 mmol), sodium iodide (117 g, 780.6mmol), copper (I) iodide (17.5 g, 91.9 mmol) and racemictrans-N,N′-dimethyl-1,2-cyclohexane diamine (20 g, 140.6 mmol) under astream of nitrogen. Purge the vessel with 3 vacuum nitrogen switches andheat to 123° C. for 18 hours. Cool to ambient temperature and filter thesolution through diatomaceous earth, and wash with ethyl acetate. Addsaturated aqueous ammonium chloride (2 L) and vigorously stir for 45minutes. Separate the layers and wash the organic layer with saturatedaqueous ammonium chloride (3×1 L), brine (300 mL), dry over magnesiumsulfate, filter, and evaporate to give a residue. Purify the residue bysilica gel chromatography eluting with 0-100% ethyl acetate iniso-hexane to give the title compound as a light yellow solid (297.9 g,95%, 81% purity). ES/MS: m/z 532.0 [M+H].

Preparation 27N-[(4aS,5S,7aS)-7a-(5-Amino-2-fluoro-phenyl)-5-(1,1-difluoroethyl)-4,4a,5,7-tetrahydrofuro[3,4-d][1,3]thiazin-2-yl]benzamide

Scheme 1, step N: CombineN-[(4aS,5S,7aS)-7a-(5-bromo-2-fluoro-phenyl)-5-(1,1-difluoroethyl)-4,4a,5,7-tetrahydrofuro[3,4-d][1,3]thiazin-2-yl]benzamide(0.372 g, 0.74 mmol) and (1R,2R)—N,N′-dimethyl-1,2-cyclohexanediamine(0.037 mL, 0.22 mmol) in ethanol (30 ml). Add sodium azide (0.194 g,2.98 mmol), followed by sodium L-ascorbate (0.66 M solution, 0.50 ml,0.33 mmol). Purge the top of the flask with nitrogen and add cupricsulfate (0.33 M solution, 0.68 ml, 0.22 mmol). Heat the reaction mixtureto 80° C. and stir for 5 hours. Cool the reaction and add cold water.Extract the mixture with ethyl acetate. Combine the organic extract anddry over sodium sulfate. Filter and concentrate under reduced pressureto give a residue. Combine the residue with palladium (10 mass % oncarbon, 0.35 g, 0.16 mmol) in ethanol (50 ml) and THF (10 ml). Purge themixture with nitrogen and with hydrogen. Stir at ambient temperatureunder 50 psi of hydrogen for 1 hour. Filter off the catalyst and washwith ethyl acetate. Concentrate the solution under reduced pressure togive a residue. Purify the residue by silica gel chromatography, elutingwith ethyl acetate:dichloromethane (0-20% gradient), to give the titlecompound (0.2184 g, 67%). ES/MS m/z 436 (M+H).

Alternate Preparation 27

Scheme 4, step B: Add 7 N ammonia in methanol (600 mL, 4.2 mol) to astirred suspension ofN-[(5S,7aS)-5-(1,1-difluoroethyl)-7a-{2-fluoro-5-[(trifluoroacetyl)amino]phenyl}-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-2-yl]benzamide(250 g, 80% purity, 376.3 mmol) in methanol (200 mL) at room temperatureand stir at ambient temperature for 18 hours. Evaporate to dryness togive the title compound as a brown gum (190 g, 375.2 mmol, 86% purity).ES/MS: m/z 436.0 [M+H].

Preparation 27b(4aS,5S,7aS)-7a-(5-Amino-2-fluorophenyl)-5-(1,1-difluoropropyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-2-amine

Scheme 1, step N: Add togetherN-[(4aS,5S,7aS)-7a-(5-bromo-2-fluorophenyl)-5-(1,1-difluoropropyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-2-yl]benzamide(0.567 g, 1.104 mmol), 1,4-dioxane (4.802 mL) and ethanol (11.04 mL),followed by sodium azide (0.2154 g, 3.313 mmol),trans-N,N′-dimethylcyclohexane-1,2-diamine (0.04760 g, 0.0528 mL, 0.3313mmol), sodium L-ascorbate 0.66 M (0.74 g, 0.74 mL, 0.4859 mmol) andwater (0.1699 mL). Finally add cupric sulphate 0.33 M (0.74 g, 0.74 mL,0.2430 mmol) and heat to 100° C. Stir overnight at 90° C. Add furthercupric sulfate 0.33 M (0.74 g, 0.74 mL, 0.2430 mmol), sodium L-ascorbate0.66 M (0.74 g, 0.74 mL, 0.4859 mmol), sodium azide (0.2154 g, 3.313mmol) and trans-N,N′-dimethylcyclohexane-1,2-diamine (0.04760 g, 0.0528mL, 0.3313 mmol). Heat the reaction at 100° C. for 1 hour, and then coolto room temperature. Pour the reaction onto brine and extract theproduct with chloroform. Filter mixture through diatomaceous earth andseparate the organics, dry over magnesium sulphate, filter, andconcentrate in vacuo. Transfer the material to a Parr flask withpalladium (5 mass %) in Lindlar's catalyst (0.113 g, 0.0533 mmol), andadd methanol (55.22 mL) under nitrogen. Hydrogenate under 276 kPahydrogen pressure with vigorous shaking for 3 hours. Add furtherpalladium (5 mass %) in Lindlar's catalyst (0.113 g, 0.0533 mmol) andhydrogenate under 345 kPa hydrogen pressure for a further 4 hours.Filter the reaction through diatomaceous earth, and wash withchloroform. Concentrate the filtrate in vacuo and then add methanol(55.22 mL) followed by lithium hydroxide hydrate (0.4634 g, 0.182 mL,11.04 mmol). Heat the reaction mixture to 70° C. for 3 hours and thencool to room temperature. Pour the reaction onto brine and extract withchloroform. Dilute the organics with methanol, and pour onto an SCX-2cartridge. Flush the cartridge with one column volume of methanol anddiscard. Then flush the SCX-2 cartridge with one column volume of 7 Mmethanolic ammonia and concentrate in vacuo to give the title compound(0.290 g, 0.840 mmol, 76%). ES/MS m/z 346 (M+H).

Preparation 27cN-[(4aS,5S,7aS)-7a-(5-Amino-2-fluoro-phenyl)-5-[cyclopropyl(difluoro)methyl]-4,4a,5,7-tetrahydrofuro[3,4-d][1,3]thiazin-2-yl]benzamide

Scheme 1, step N: CombineN-[(4aS,5S,7aS)-7a-(5-bromo-2-fluoro-phenyl)-5-[cyclopropyl(difluoro)methyl]-4,4a,5,7-tetrahydrofuro[3,4-d][1,3]thiazin-2-yl]benzamide(31 mg, 0.059 mmol) and (1R,2R)—N,N′-dimethyl-1,2-cyclohexanediamine(0.0049 mL, 0.030 mmol) in ethanol (3 ml). Add sodium azide (31 mg, 0.47mmol), followed by sodium L-ascorbate (0.66 M solution, 0.089 ml, 0.059mmol). Purge the top of the flask with nitrogen and add cupric sulfate(0.33 M solution, 0.18 ml, 0.059 mmol). Heat the reaction mixture to 80°C. and stir for 3 hours. Cool the reaction and add cold water. Extractthe mixture with ethyl acetate. Combine the organic extract and dry oversodium sulfate. Filter and concentrate under reduced pressure to give aresidue. Combine the residue with palladium (10 mass % on carbon, 30 mg,0.014 mmol) in ethanol (20 ml) and THF (5 ml). Purge the mixture withnitrogen and with hydrogen. Stir at ambient temperature under hydrogenat 40 psi for 4 hours. Filter off the catalyst and wash with ethylacetate. Concentrate the solution under reduced pressure to give aresidue. Purify the residue by silica gel chromatography, eluting withethyl acetate:hexanes (0-100% gradient), to give the title compound (21mg, 77%). ES/MS m/z 462 (M+H).

Preparation 28 (4aS,5S,7aS)-7a-(5-Amino-2-fluorophenyl)-5-(1,1-difluoroethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-2-amine

Scheme 4, step B: DissolveN-[(4aS,5S,7aS)-7a-(5-amino-2-fluoro-phenyl)-5-(1,1-difluoroethyl)-4,4a,5,7-tetrahydrofuro[3,4-d][1,3]thiazin-2-yl]benzamide(216.4 g, 88% purity, 435.9 mmol) in pyridine (400 mL), ethanol (100 mL)and THF (300 mL). Add O-methylhydroxylamine hydrochloride (190 g, 2275.0mmol) and stir at ambient temperature for 18 hours. Dilute with2-methyltetrahydrofuran (1 L) and wash with water (2×300 mL). Isolatethe organic layer and add 35% aqueous ammonium hydroxide (100 mL) to theaqueous. Extract with 2-methyltetrahydrofuran (300 mL) then saturatewith sodium chloride and extract with 2-methyltetrahydrofuran (2×300mL). Combine the organic extracts, wash with brine (300 mL) andevaporate to a residue. Dissolve in methanol (200 mL), add 7 N ammoniain methanol (100 mL, 700 mmol) and stir at room temperature for 18hours. Further ammonia can be added if any trifluoroacetamide impurityremains. Remove the solvent under reduced pressure and dissolve theresidue in aqueous 2 N aqueous hydrochloric acid (1.5 L). Extract withdichloromethane (6×500 mL), combine the organic layers and remove thesolvent under reduced pressure to a total volume of about 1 L. Wash with2 N aqueous hydrochloric acid (300 mL) and combine all aqueous washings.Add 2-methyltetrahydrofuran (1 L) and stir vigorously while adjustingthe pH to basic with sodium bicarbonate until no gas evolution isobserved. Separate the layers and extract the aqueous with2-methyltetrahydrofuran (2×500 mL). Dry the combined organic extractswith magnesium sulfate, filter, and evaporate to give a brown solid.Purify the residue by silica gel chromatography eluting with 0-100%dichloromethane in THF. Evaporate the product containing fractions withethyl acetate/heptane to give the title compound as a fine beige powder(106 g, 70%, 95% purity). ES/MS: m/z 332.0 [M+H], [α]_(D) ²⁰=+42.11°(C=0.532, chloroform).

Preparation 29 5-(1H-1,2,4-Triazol-1-yl)pyrazine-2-carboxylic acid

Stir a mixture of methyl 5-chloropyrazine-2-carboxylate (124 g, 718.55mmol), 1H-1,2,4-triazole (198.5 g, 2874.2 mmol) and potassium carbonate(297.92 g, 2155.6 mmol) in N,N-dimethylformamide (1 L) at 100° C. for 15hours. Cool to ambient temperature and pour into water (2 L). Adjust thepH of the solution to 2-3 using concentrated aqueous hydrochloric acid(about 500 mL) and stir for 30 minutes. Collect the resulting solid byfiltration and wash with water. Add water (500 mL) and ethanol (500 mL),heat to 50-60° C. for 4 hours, and cool to ambient temperature. Collectthe solids by filtration and dry under vacuum at 40° C. to give thetitle compound as a white solid. ES/MS: m/z 190.0 (M−H).

Preparation 30 N-[3-[(4aS,5S,7aS)-2-Benzamido-5-(1,1-difluoroethyl)-4,4a,5,7-tetrahydrofuro[3,4-d][1,3]thiazin-7a-yl]-4-fluoro-phenyl]-5-(1,2,4-triazol-1-yl)pyrazine-2-carboxamide

Scheme 3, step A: Add togetherN-[(4as,5s,7as)-7a-(5-amino-2-fluoro-phenyl)-5-(1,1-difluoroethyl)-4,4a,5,7-tetrahydrofuro[3,4-d][1,3]thiazin-2-yl]benzamide(0.139 g, 0.32 mmol), 5-(1H-1,2,4-triazol-1-yl)pyrazine-2-carboxylicacid (0.0852 g, 0.45 mmol), and HOAt (0.0575 g, 0.41 mmol) indichloromethane (4 ml):dimethylformamide (1 mL). AddN,N-diisopropylethylamine (0.11 mL, 0.63 mmol) to the solution followedby EDCI (0.079 g, 0.41 mmol) in one portion. Stir the reaction mixtureat ambient temperature for 18 hours. Dilute the solution with ethylacetate, wash with water and brine, and separate the phases. Extractwith ethyl acetate. Combine the organic extract and dry over magnesiumsulfate. Filter the solution and concentrate under reduced pressure togive a residue. Purify the residue by silica gel chromatography, elutingwith ethyl acetate:dichloromethane (0-30% gradient), to give the titlecompound (0.1140 g, 59%). ES/MS m/z 609 (M+H).

Preparation 30aN-[3-[(4aS,5S,7aS)-2-Benzamido-5-[cyclopropyl(difluoro)methyl]-4,4a,5,7-tetrahydrofuro[3,4-d][1,3]thiazin-7a-yl]-4-fluoro-phenyl]-5-(1,2,4-triazol-1-yl)pyrazine-2-carboxamide

Scheme 3, step A: Add togetherN-[(4aS,5S,7aS)-7a-(5-amino-2-fluoro-phenyl)-5-[cyclopropyl(difluoro)methyl]-4,4a,5,7-tetrahydrofuro[3,4-d][1,3]thiazin-2-yl]benzamide(21 mg, 0.045 mmol), 5-(1H-1,2,4-triazol-1-yl)pyrazine-2-carboxylic acid(10 mg, 0.054 mmol), and HOBT (10 mg, 0.059 mmol) in dichloromethane(2.5 ml):dimethylformamide (0.5 mL). Add N,N-diisopropylethylamine(0.016 mL, 0.091 mmol) to the solution followed by EDCI (11 mg, 0.059mmol) in one portion. Stir the reaction mixture at ambient temperaturefor 18 hours. Dilute the solution with ethyl acetate, water, and 1 NNaOH (0.5 mL) and extract with ethyl acetate (3×). Combine the organicextracts and dry over sodium sulfate. Filter the solution andconcentrate under reduced pressure to give a residue. Purify the residueby silica gel chromatography, eluting with ethyl acetate:dichloromethane(0-100% gradient), to give the title compound (20 mg, 69%). ES/MS m/z635 (M+H).

Example 1N-[3-[(4aS,5S,7aS)-2-Amino-5-(1,1-difluoroethyl)-4,4a,5,7-tetrahydrofuro[3,4-d][1,3]thiazin-7a-yl]-4-fluoro-phenyl]-5-(1,2,4-triazol-1-yl)pyrazine-2-carboxamide

Scheme 3, step B: Heat a mixture ofN-[3-[(4aS,5S,7aS)-2-benzamido-5-(1,1-difluoroethyl)-4,4a,5,7-tetrahydrofuro[3,4-d][1,3]thiazin-7a-yl]-4-fluoro-phenyl]-5-(1,2,4-triazol-1-yl)pyrazine-2-carboxamide(0.1148 g, 0.189 mmol), O-methylhydroxylamine hydrochloride (0.1575 g,1.886 mmol), and pyridine (0.15 ml, 1.886 mmol) in THF (2 mL) andethanol (2 mL) at 45° C. for 5 hours. Cool the reaction mixture toambient temperature and stir for 2 days. Concentrate the solution underreduced pressure to give a residue. Purify the residue by silica gelchromatography, eluting with 7 N NH₃ in methanol:dichloromethane (0-3%gradient), to give the title compound (0.086 g, 90%). ES/MS m/z 505(M+H).

Alternative Preparation Example 1

Scheme 4 Step D: Stir(4aS,5S,7aS)-7a-(5-amino-2-fluorophenyl)-5-(1,1-difluoroethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-2-amine(96.5 g, 291 mmol) in ethyl acetate (1 L) under a nitrogen atmosphere at50° C. and add 5-(1H-1,2,4-triazol-1-yl)pyrazine-2-carboxylic acid (84g, 439.45 mmol) slowly to the warm solution. Stir for 10 minutes and addT3P® (1.67 M in ethyl acetate, 350 mL, 585 mmol) and stir at 50° C. for17 hours. Cool to ambient temperature, dilute with dichloromethane (1 L)and stir while quenching with a solution of sodium carbonate in water(128 g, 1.21 mol in 1 L). Dilute with dichloromethane (1 L) and water (2L) and stir vigorously for 1 hour. Filter through diatomaceous earth andwash with dichloromethane (3×500 mL), methanol (500 mL), water (500 mL),aqueous saturated sodium bicarbonate (500 mL), and 1:1methanol:dichloromethane (6×500 mL). Separate the layers and extract theaqueous with dichloromethane (3×1 L). Combine all organic phases andevaporate to give a residue. Sonicate the residue in dichloromethane (1L) for 15 minutes and collect the solids by filtration washing withdichloromethane (5×200 mL). Add saturated aqueous sodium hydrogencarbonate until pH 8 is obtained and stir vigorously withdichloromethane (1 L) and methanol (500 mL). Remove the solids byfiltration and extract the filtrate with dichloromethane (2×500 mL).Dissolve the solids with dichloromethane:methanol (1:1, 500 mL) andcombine this solution with the other organic phases. Remove the solventunder reduced pressure adding dichloromethane to maintain a solution andthen once a final volume of about 300 mL is obtained, purify thesolution by silica gel chromatography, eluting with 5% of 0.3 Mammonia/methanol in dichloromethane to give a light brown solid.Dissolve the solid in hot ethanol (2.5 L), filter while hot, and cool toambient temperature over 1 hour. Collect the solids by filtration andwash with ethanol (2×250 mL) and dry under vacuum. Evaporate thefiltrate to dryness and further purify by silica gel chromatographyeluting first with 65% ethyl acetate in 50:1 iso-hexane/7 N ammonia inmethanol then 50:1 ethyl acetate/7 N ammonia in methanol. If required,further purification can be completed by SFC, column: Chiralpak AD-H(5μ), 50×250 mm; eluent: 35% isopropanol (0.2% diethylmethylamine) inCO₂; flow rate: 300 g/minute at UV 220 nm. After evaporation and vacuumdrying, slurry the material in ethanol (1.5 L) and stir with gentlewarming between (36 and 45° C.) for 20 minutes. Collect the solid byfiltration washing with ethanol (100 mL). Further material can berecovered from the filtrate; evaporate to dryness, reflux in ethanol,remove the solids by hot filtration and then cool the filtrate toambient temperature. Collect solids by filtration, washing with ethanoland combine with the material obtained from the above filtration. Drythe combined solids under vacuum at 40° C. to give the title compound asa white solid (103.3 g, 68%, containing 2.5 wt % ethanol). ES/MS m/z505.0 (M+H), [α]_(D) ²⁰=+149.4° (C=1, chloroform).

Example 1AN-[3-[(4aS,5S,7aS)-2-amino-5-(1,1-difluoroethyl)-4,4a,5,7-tetrahydrofuro[3,4-d][1,3]thiazin-7a-yl]-4-fluoro-phenyl]-5-(1,2,4-triazol-1-yl)pyrazine-2-carboxamide4-methylbenzenesulfonate

DissolveN-[3-[(4aS,5S,7aS)-2-amino-5-(1,1-difluoroethyl)-4,4a,5,7-tetrahydrofuro[3,4-d][1,3]thiazin-7a-yl]-4-fluoro-phenyl]-5-(1,2,4-triazol-1-yl)pyrazine-2-carboxamide(600 mg, 1.189 mmol) in acetone (9 mL) and water (1 mL). Heat theresulting suspension to 60° C. Add p-toluenesulfonic acid monohydrate(420 mg, 2.208 mmol) dissolved in acetone (1 mL). Stir the mixtureovernight at 60° C. Cool the mixture to room temperature, filter thesolids by vacuum and wash with acetone (1 mL) and air dry overnight togive the title compound (743 mg, 73%).

X-Ray Powder Diffraction (XRD)

The XRD patterns of crystalline solids are obtained on a Bruker D4Endeavor X-ray powder diffractometer, equipped with a CuKa sourceλ=1.54060 Å and a Vantec detector, operating at 35 kV and 50 mA. Thesample is scanned between 4 and 40° in 2θ, with a step size of 0.009° in2θ, a scan rate of 0.5 seconds/step, with 0.6 mm divergence, 5.28 fixedanti-scatter, and 9.5 mm detector slits. The dry powder is packed on aquartz sample holder and a smooth surface is obtained using a glassslide. The crystal form diffraction patterns are collected at ambienttemperature and relative humidity. It is well known in thecrystallography art that, for any given crystal form, the relativeintensities of the diffraction peaks may vary due to preferredorientation resulting from factors such as crystal morphology and habit.Where the effects of preferred orientation are present, peak intensitiesare altered, but the characteristic peak positions of the polymorph areunchanged. See, e.g., The United States Pharmacopeia #23, NationalFormulary #18, pages 1843-1844, 1995. Furthermore, it is also well knownin the crystallography art that for any given crystal form the angularpeak positions may vary slightly. For example, peak positions can shiftdue to a variation in the temperature or humidity at which a sample isanalyzed, sample displacement, or the presence or absence of an internalstandard. In the present case, a peak position variability of ±0.2 in 20will take into account these potential variations without hindering theunequivocal identification of the indicated crystal form. Confirmationof a crystal form may be made based on any unique combination ofdistinguishing peaks (in units of ° 2θ), typically the more prominentpeaks. The crystal form diffraction patterns, collected at ambienttemperature and relative humidity, were adjusted based on NIST 675standard peaks at 8.853 and 26.774 degrees 2-theta.

A prepared sample of crystallineN-[3-[(4aS,5S,7aS)-2-amino-5-(1,1-difluoroethyl)-4,4a,5,7-tetrahydrofuro[3,4-d][1,3]thiazin-7a-yl]-4-fluoro-phenyl]-5-(1,2,4-triazol-1-yl)pyrazine-2-carboxamide4-methylbenzenesulfonate is characterized by an XRD pattern using CuKaradiation as having diffraction peaks (2-theta values) as described inthe Table below. Specifically, the pattern contains a peak at 17.3° incombination with one or more of the peaks selected from the groupconsisting of 14.8, 12.7, and 4.9; with a tolerance for the diffractionangles of 0.2 degrees.

TABLE 1 X-ray powder diffraction peaks of Example 1A. Angle RelativeIntensity Peak (°2-Theta +/− 0.2°) (% of most intense peak) 1 4.9 48 29.4 14 3 12.7 52 4 14.8 60 5 17.3 100 6 19.8 44 7 24.9 35 8 25.3 37 926.8 19 10 28.2 14

Example 1BN-[3-[(4aS,5S,7aS)-2-amino-5-(1,1-difluoroethyl)-4,4a,5,7-tetrahydrofuro[3,4-d][1,3]thiazin-7a-yl]-4-fluoro-phenyl]-5-(1,2,4-triazol-1-yl)pyrazine-2-carboxamidemalonate

Add togetherN-[3-[(4aS,5S,7aS)-2-amino-5-(1,1-difluoroethyl)-4,4a,5,7-tetrahydrofuro[3,4-d][1,3]thiazin-7a-yl]-4-fluoro-phenyl]-5-(1,2,4-triazol-1-yl)pyrazine-2-carboxamide(201 mg, 0.398 mmol) and malonic acid (104 mg, 0.999 mmol) in 95%ethanol-water (15 mL). Stir the mixture 65° C. until a solution a clearsolution is obtained. A thick white solid precipitates after a fewminutes. Stir the suspension for 1 hour at 55° C. and then cool to roomtemperature with stirring. Filter the solids under vacuum and air dryfor 2 days to give the title compound (477 mg, 80%).

A prepared sample of crystallineN-[3-[(4aS,5S,7aS)-2-amino-5-(1,1-difluoroethyl)-4,4a,5,7-tetrahydrofuro[3,4-d][1,3]thiazin-7a-yl]-4-fluoro-phenyl]-5-(1,2,4-triazol-1-yl)pyrazine-2-carboxamidemalonate is characterized by an XRD pattern using CuKa radiation ashaving diffraction peaks (2-theta values) as described in Table 2 below.Specifically, the pattern contains a peak at 22.7 in combination withone or more of the peaks selected from the group consisting of 16.8,17.2, and 24.0; with a tolerance for the diffraction angles of 0.2degrees.

TABLE 2 X-ray powder diffraction peaks of Example 1B. Angle RelativeIntensity Peak (°2-Theta +/− 0.2°) (% of most intense peak) 1 5.5 39 210.3 44 3 11.8 55 4 15.3 39 5 16.8 62 6 17.2 57 7 18.3 41 8 22.4 60 922.7 100 10 24.0 53

Example 1CN-[3-[(4aS,5S,7aS)-2-amino-5-(1,1-difluoroethyl)-4,4a,5,7-tetrahydrofuro[3,4-d][1,3]thiazin-7a-yl]-4-fluoro-phenyl]-5-(1,2,4-triazol-1-yl)pyrazine-2-carboxamidehydrate

SuspendN-[3-[(4aS,5S,7aS)-2-amino-5-(1,1-difluoroethyl)-4,4a,5,7-tetrahydrofuro[3,4-d][1,3]thiazin-7a-yl]-4-fluoro-phenyl]-5-(1,2,4-triazol-1-yl)pyrazine-2-carboxamide(116 mg, 0.23 mmol) in 1:1 THF:water (2 mL) at 70° C. Stir the solutionfor at least 2 days, filter the solid, and dry under a nitrogen streamto give the title compound.

A prepared sample ofN-[3-[(4aS,5S,7aS)-2-amino-5-(1,1-difluoroethyl)-4,4a,5,7-tetrahydrofuro[3,4-d][1,3]thiazin-7a-yl]-4-fluoro-phenyl]-5-(1,2,4-triazol-1-yl)pyrazine-2-carboxamidehydrate is characterized by an XRD pattern using CuKa radiation ashaving diffraction peaks (2-theta values) as described in Table 3 below.Specifically, the pattern contains a peak at 13.0 in combination withone or more of the peaks selected from the group consisting of 7.8,10.5, 11.0, 14.9, 19.7, 21.3, and 26.9 with a tolerance for thediffraction angles of 0.2 degrees.

TABLE 3 X-ray powder diffraction peaks of Example 1C. Angle RelativeIntensity Peak (°2-Theta +/− 0.2°) (% of most intense peak) 1 7.8 82 210.5 68 3 11.0 38 4 13.0 100 5 13.2 48 6 14.9 44 7 16.6 30 8 19.1 32 919.7 93 10 21.1 29 11 21.3 73 12 22.0 26 13 22.3 52 14 26.9 65

Example 2N-[3-[(4aS,5S,7aS)-2-Amino-5-(1,1-difluoropropyl)-4,4a,5,7-tetrahydrofuro[3,4-d][1,3]thiazin-7a-yl]-4-fluoro-phenyl]-5-(1,2,4-triazol-1-yl)pyrazine-2-carboxamide

Scheme 3, steps A and B: Add together5-(1H-1,2,4-triazol-1-yl)pyrazine-2-carboxylic acid (0.116 g, 0.608mmol), acetonitrile (4.05 mL), dimethylformamide (0.00314 mL), and thenoxalyl chloride (0.154 g, 0.105 mL, 1.22 mmol) dropwise under nitrogen.Stir the reaction for 30 minutes and then concentrate in vacuo. Dissolvethe residue in acetonitrile (4.05 mL,) and add dropwise to a mixture of(4aS,5S,7aS)-7a-(5-amino-2-fluorophenyl)-5-(1,1-difluoropropyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-2-amine(0.140 g, 0.405 mmol) in ethanol (4.05 mL) and water (1.35 mL) withstirring. Upon complete addition, dilute the reaction with chloroformand wash with saturated sodium bicarbonate solution. Dilute the organicswith methanol and add to an SCX-2 cartridge. Flush the SCX-2 cartridgewith one column volume of methanol and discard, and then flush the SCX-2cartridge with one column volume of 7 M methanolic ammonia. Concentratethe methanolic ammonia flush in vacuo and purify by silica gelchromatography, eluting with dichloromethane/methanol (100-85% gradient)to give a residue. Further purify by achiral SFC (Supercritical FluidChromatography) (Column: benzenesulfonamide (BzS) (5μ), PrincetonChromatography, 21.2×250 mm; eluent: 22% methanol (1% 2 M ammonia inmethanol) in CO₂; flow rate: 70 mL/minute at UV 250 nm; back pressure:100 bar; temperature: 40° C.). Solubilize the residue in chloroform andwash with brine. Pass the organics through a hydrophobic frit andconcentrate in vacuo to give the title compound (0.0658 g, 0.127 mmol,31%). ES/MS m/z 519 (M+H).

Example 3N-[3-[(4aS,5S,7aS)-2-Amino-5-[cyclopropyl(difluoro)methyl]-4,4a,5,7-tetrahydrofuro[3,4-d][1,3]thiazin-7a-yl]-4-fluoro-phenyl]-5-(1,2,4-triazol-1-yl)pyrazine-2-carboxamide

Scheme 3, step B: Heat a mixture ofN-[3-[(4aS,5S,7aS)-2-benzamido-5-[cyclopropyl(difluoro)methyl]-4,4a,5,7-tetrahydrofuro[3,4-d][1,3]thiazin-7a-yl]-4-fluoro-phenyl]-5-(1,2,4-triazol-1-yl)pyrazine-2-carboxamide(20 mg, 0.0315 mmol), O-methylhydroxylamine hydrochloride (26 mg, 0.315mmol), and pyridine (0.026 ml, 0.315 mmol) in ethanol (3 mL) at 55° C.for 18 hours. Cool the reaction mixture to ambient temperature andconcentrate the solution under reduced pressure to give a residue.Purify the residue by silica gel chromatography, eluting with 7 N NH₃ inmethanol: dichloromethane (0.5-10% gradient), to give the title compound(15 mg, 90%). ES/MS m/z 531 (M+H).

In Vitro Assay Procedures:

To assess selectivity of BACE1 over BACE2, the test compound isevaluated in FRET-based enzymatic assays using specific substrates forBACE1 and BACE2 as described below. For in vitro enzymatic and cellularassays, the test compound is prepared in DMSO to make up a 10 mM stocksolution. The stock solution is serially diluted in DMSO to obtain aten-point dilution curve with final compound concentrations ranging from10 μM to 0.05 nM in a 96-well round-bottom plate before conducting thein vitro enzymatic and whole cell assays.

In Vitro Protease Inhibition Assays:

Expression of huBACE1:Fc and huBACE2:Fc

Human BACE1 (accession number: AF190725) and human BACE2 (accessionnumber: AF 204944) are cloned from total brain cDNA by RT-PCR. Thenucleotide sequences corresponding to amino acid sequences #1 to 460 areinserted into the cDNA encoding human IgG₁ (Fc) polypeptide (Vassar etal., Science, 286, 735-742 (1999)). This fusion protein of BACE1(1-460)or BACE2(1-460) and human Fc, named huBACE1:Fc and huBACE2:Fcrespectively, are constructed into the pJB02 vector. HumanBACE1(1-460):Fc (huBACE1:Fc) and human BACE2(1-460):Fc (huBACE2:Fc) aretransiently expressed in HEK293 cells. 250 μg cDNA of each construct aremixed with Fugene 6 and added to 1 liter HEK293 cells. Four days afterthe transfection, conditioned media are harvested for purification.huBACE1:Fc and huBACE2:Fc are purified by Protein A chromatography asdescribed below. The enzymes are stored at −80° C. in small aliquots.(See Yang, et. al., J. Neurochemistry, 91(6) 1249-59 (2004).

Purification of huBACE1:Fc and huBACE2:Fc

Conditioned media of HEK293 cell transiently transfected with huBACE1:Fcor huBACE2:Fc cDNA are collected. Cell debris is removed by filteringthe conditioned media through 0.22 μm sterile filter. 5 ml ProteinA-agarose (bed volume) is added to 4 liter conditioned media. Thismixture is gently stirred overnight at 4° C. The Protein A-agarose resinis collected and packed into a low-pressure chromatography column. Thecolumn is washed with 20× bed volumes of PBS at a flow rate 20 ml perhour. Bound huBACE1:Fc or huBACE2:Fc protein is eluted with 50 mM aceticacid, pH 3.6, at flow rate 20 ml per hour. 1 ml fractions of eluent areneutralized immediately with 0.5 ml 200 mM ammonium acetate, pH 6.5. Thepurity of final product is assessed by electrophoresis in 4-20%Tris-Glycine SDS-PAGE. The enzyme is stored at −80° C. in smallaliquots.

BACE1 FRET Assay

Serial dilutions of the test compound are prepared as described above.The compound is further diluted 20× in KH₂PO₄ buffer. Ten μL of eachdilution is added to each well on row A to H of a corresponding lowprotein binding black plate containing the reaction mixture (25 μL of 50mM KH₂PO₄, pH 4.6, 1 mM TRITON® X-100, 1 mg/mL BSA, and 15 μM of FRETsubstrate based upon the sequence of APP) (See Yang, et. al., J.Neurochemistry, 91(6) 1249-59 (2004)). The content is mixed well on aplate shaker for 10 minutes. Fifteen μL of two hundred μM humanBACE1(1-460):Fc (See Vasser, et al., Science, 286, 735-741 (1999)) inthe KH₂PO₄ buffer is added to the plate containing substrate and thetest compound to initiate the reaction. The RFU of the mixture at time 0is recorded at excitation wavelength 355 nm and emission wavelength 460nm, after brief mixing on a plate shaker. The reaction plate is coveredwith aluminum foil and kept in a dark humidified oven at roomtemperature for 16 to 24 hours. The RFU at the end of incubation isrecorded with the same excitation and emission settings used at time 0.The difference of the RFU at time 0 and the end of incubation isrepresentative of the activity of BACE1 under the compound treatment.RFU differences are plotted versus inhibitor concentration and a curveis fitted with a four-parameter logistic equation to obtain the IC₅₀value. (May, et al., Journal of Neuroscience, 31, 16507-16516 (2011)).

The compound of Example 1 herein is tested essentially as describedabove and exhibits an IC₅₀ for BACE1 of 1.19 nM±0.48, n=4 (Mean±SEM;SEM=standard error of the mean). This data demonstrates that thecompound of Example 1 inhibits purified recombinant BACE1 enzymeactivity in vitro.

BACE2 TMEM27 FRET Assay

Transmembrane protein 27 (TMEM27) (Accession Number NM_020665), alsoknown as Collectrin) is a recently described substrate for BACE2, butnot BACE1 (Esterhazy, et al, Cell Metabolism, 14, 365-377 (2011)). Toevaluate the test compound for inhibition of BACE2 enzymatic activity, aFRET peptide (dabcyl-QTLEFLKIPS-LucY) based upon the amino acid sequenceof human TMEM27 is used as a substrate (Esterhazy, et al, CellMetabolism, 14, 365-377 (2011)). Serial dilutions of the test compoundare prepared as described above. The compound is further diluted 20× inKH₂PO₄ buffer. Ten μL of each dilution is added to each well on row A toH of a corresponding low protein binding black plate containing thereaction mixture (25 μL of 50 mM KH₂PO₄, pH 4.6, 1 mM TRITON® X-100, 1mg/mL BSA, and 5 μM of TMEM FRET substrate). Fifteen μL of twenty μMhuman BACE2 (1-460):Fc (See Vasser, et al., Science, 286, 735-741(1999)) in KH₂PO₄ buffer is then added to the plate containing substrateand the test compound to initiate the reaction. The content is mixedwell on a plate shaker for 10 minutes. The RFU of the mixture at time 0is recorded at excitation wavelength 430 nm and emission wavelength 535nm. The reaction plate is covered with aluminum foil and kept in a darkhumidified oven at room temperature for 16 to 24 hours. The RFU at theend of incubation is recorded with the same excitation and emissionsettings used at time 0. The difference of the RFU at time 0 and the endof incubation is representative of the activity of BACE2 under thecompound treatment. RFU differences are plotted versus inhibitorconcentration and a curve is fitted with a four-parameter logisticequation to obtain the IC₅₀ value. (May, et al., Journal ofNeuroscience, 31, 16507-16516 (2011)).

The compound of Example 1 herein is tested essentially as describedabove and exhibits a BACE2 IC₅₀ of 479 nM±202, n=4 (Mean±SEM;SEM=standard error of the mean). The ratio of BACE1 (FRET IC₅₀ enzymeassay) to BACE2 (TMEM27 FRET IC₅₀ assay) is about 400-fold, indicatingfunctional selectivity for inhibiting the BACE1 enzyme. The data setforth above demonstrates that the compound of Example 1 is selective forBACE1 over BACE2.

SH-SY5YAPP695Wt Whole Cell Assay

The routine whole cell assay for the measurement of inhibition of BACE1activity utilizes the human neuroblastoma cell line SH-SY5Y (ATCCAccession No. CRL2266) stably expressing a human APP695Wt cDNA. Cellsare routinely used up to passage number 6 and then discarded.

SH-SY5YAPP695Wt cells are plated in 96 well tissue culture plates at5.0×10⁴ cells/well in 200 μL culture media (50% MEM/EBSS and Ham's F12,lx each sodium pyruvate, non-essential amino acids and Na bicarbonatecontaining 10% FBS). The following day, media is removed from the cells,fresh media added then incubated at 37° C. for 24 hours in thepresence/absence of test compound at the desired concentration range.

At the end of the incubation, conditioned media are analyzed forevidence of beta-secretase activity by analysis of Abeta peptides 1-40and 1-42 by specific sandwich ELISAs. To measure these specific isoformsof Abeta, monoclonal 2G3 is used as a capture antibody for Abeta 1-40and monoclonal 21F12 as a capture antibody for Abeta 1-42. Both Abeta1-40 and Abeta 1-42 ELISAs use biotinylated 3D6 as the reportingantibody (for description of antibodies, see Johnson-Wood, et al., Proc.Natl. Acad. Sci. USA 94, 1550-1555 (1997)). The concentration of Abetareleased in the conditioned media following the compound treatmentcorresponds to the activity of BACE1 under such conditions. The 10-pointinhibition curve is plotted and fitted with the four-parameter logisticequation to obtain the IC₅₀ values for the Abeta-lowering effect. Thecompound of Example 1 is tested essentially as described above andexhibits the following activity for Abeta-lowering as shown in table 4.

TABLE 4 SH-SY5YAPP695Wt SH-SY5YAPP695Wt A-beta (1-40) ELISA A-beta(1-42) ELISA Example # IC₅₀ (nM) IC₅₀ (nM) 1 0.385 ± 0.163, n = 4 0.381± 0.266, n = 4 (Mean ± SEM; SEM = standard error of the mean)

In Vivo Inhibition of Beta-Secretase

Several animal models, including mouse, guinea pig, dog, and monkey, maybe used to screen for inhibition of beta-secretase activity in vivofollowing compound treatment. Animals used in this invention can be wildtype, transgenic, or gene knockout animals. For example, the PDAPP mousemodel, prepared as described in Games et al., Nature 373, 523-527(1995), and other non-transgenic or gene knockout animals are useful toanalyze in vivo inhibition of Abeta and sAPPbeta production in thepresence of inhibitory compounds. Generally, 2 month old PDAPP mice,gene knockout mice or non-transgenic animals are administered compoundformulated in vehicles, such as corn oil, beta-cyclodextran, phosphatebuffers, PHARMASOLVE®, or other suitable vehicles via oral,subcutaneous, intra-venous, feeding, or other route of administration.One to twenty-four hours following the administration of compound,animals are sacrificed, and brains are removed for analysis of Abeta1-x. “Abeta 1-x” as used herein refers to the sum of Abeta species thatbegin with residue 1 and end with a C-terminus greater than residue 28.This detects the majority of Abeta species and is often called “totalAbeta”. Total Abeta peptides (Abeta 1-x) levels are measured by asandwich ELISA, using monoclonal 266 as a capture antibody andbiotinylated 3D6 as reporting antibody. (See May, et al., Journal ofNeuroscience, 31, 16507-16516 (2011)).

For acute studies, compound or appropriate vehicle is administered andanimals are sacrificed at about 3 hours after dosing. Brain tissue, isobtained from selected animals and analyzed for the presence of Abeta1-x. After chronic dosing brain tissues of older APP transgenic animalsmay also be analyzed for the amount of beta-amyloid plaques followingcompound treatment.

Animals (PDAPP or other APP transgenic or non-transgenic mice)administered an inhibitory compound may demonstrate the reduction ofAbeta in brain tissues, as compared with vehicle-treated controls ortime zero controls. For example, a 3, 10, and 30 mg/kg oral dose ofExample 1, to young female PDAPP mice reduced Abeta 1-x peptide levelsin brain hippocampus by 23% (non-significant), 43% (p<0.05), and 58%(p<0.01), respectively. In brain cortical tissue, doses of 3, 10, and 30mg/kg of Example 1 reduced Abeta 1-x levels by 43%, 59%, and 73% (allvalues p<0.01) compared to vehicle-treated mice three hours afterdosing.

Given the activity of the Example 1, against the BACE1 enzyme in vitro,these Abeta-lowering effects are consistent with BACE inhibition invivo, and further demonstrate CNS penetration of Example 1.

These studies show that compound of the present invention inhibits BACE1and is, therefore, useful in reducing Abeta levels.

We claim:
 1. A compound of the formula:

wherein R is methyl, ethyl, or cyclopropyl; or a pharmaceuticallyacceptable salt thereof.
 2. The compound or salt thereof according toclaim 1 wherein R is methyl.
 3. The compound or salt thereof accordingto claim 2 wherein the compound is:


4. The compound or salt thereof according to claim 3 wherein thecompound is:


5. The compound according to claim 4 which is:


6. The salt according to claim 4 which is:


7. The salt according to claim 4 which is:


8. The compound according to claim 4 which is:


9. The compound or salt thereof according to claim 4 wherein thecompound isN-[3-[(4aS,5S,7aS)-2-amino-5-(1,1-difluoroethyl)-4,4a,5,7-tetrahydrofuro[3,4-d][1,3]thiazin-7a-yl]-4-fluoro-phenyl]-5-(1,2,4-triazol-1-yl)pyrazine-2-carboxamide.10. The compound according to claim 5 which isN-[3-[(4aS,5S,7aS)-2-amino-5-(1,1-difluoroethyl)-4,4a,5,7-tetrahydrofuro[3,4-d][1,3]thiazin-7a-yl]-4-fluoro-phenyl]-5-(1,2,4-triazol-1-yl)pyrazine-2-carboxamide.11. The compound according to claim 4 which isN-[3-[(4aS,5S,7aS)-2-amino-5-(1,1-difluoroethyl)-4,4a,5,7-tetrahydrofuro[3,4-d][1,3]thiazin-7a-yl]-4-fluoro-phenyl]-5-(1,2,4-triazol-1-yl)pyrazine-2-carboxamidehydrate.
 12. The salt according to claim 4 which isN-[3-[(4aS,5S,7aS)-2-amino-5-(1,1-difluoroethyl)-4,4a,5,7-tetrahydrofuro[3,4-d][1,3]thiazin-7a-yl]-4-fluoro-phenyl]-5-(1,2,4-triazol-1-yl)pyrazine-2-carboxamide4-methylbenzenesulfonate.
 13. The salt according to claim 4 which isN-[3-[(4aS,5S,7aS)-2-amino-5-(1,1-difluoroethyl)-4,4a,5,7-tetrahydrofuro[3,4-d][1,3]thiazin-7a-yl]-4-fluoro-phenyl]-5-(1,2,4-triazol-1-yl)pyrazine-2-carboxamidemalonate.
 14. A method of treating Alzheimer's disease in a patient,comprising administering to a patient in need of such treatment aneffective amount of a compound of claim 1, or a pharmaceuticallyacceptable salt thereof.
 15. A method of treating the progression ofmild cognitive impairment to Alzheimer's disease in a patient,comprising administering to a patient in need of such treatment aneffective amount of a compound of claim 1, or a pharmaceuticallyacceptable salt thereof.
 16. A pharmaceutical composition, comprising acompound or a pharmaceutically acceptable salt thereof according toclaim 1 with one or more pharmaceutically acceptable carriers, diluents,or excipients.
 17. A process for preparing a pharmaceutical composition,comprising admixing a compound or a pharmaceutically acceptable saltthereof according to claim 1 with one or more pharmaceuticallyacceptable carriers, diluents, or excipients.